Escape from the Ponto-Caspian: evolution and biogeography of an endemic goby species flock (Benthophilinae: Gobiidae: Teleostei

Carol A . Stepien

Endemic Ponto-Caspian gobies include a flock of 24 ‘‘neogobiin” species (containing the nominal genera and subgenera Apollonia, Babka, Neogobius, Mesogobius, Ponticola, and Proterorhinus; Teleostei: Gobiidae), of which a large proportion (5 species; 21%) recently escaped to invade other freshwater Eurasian systems and the North American Great Lakes. We provide its first comprehensive phylogenetic and biogeographic analysis based on 4709 bp sequences from two mitochondrial and two nuclear genes with maximum parsimony, likelihood, and Bayesian approaches. We additionally compare its relationships with the tadpole gobies (Benthophilus and Caspiosoma), which comprise a related endemic Ponto-Caspian gobiid group; along with a variety of postulated relatives and outgroups. Results of all phylogenetic approaches are highly congruent and provide very strong support for recognizing the subfamily Benthophilinae; which encompasses both the ‘‘neogobiins” and tadpole gobies, and genetically diverges from other Gobiidae subfamilies—including (non-monophyletic) Gobiinae and Gobinellinae. Benthophilinae contains three tribes: Neogobiini (Neogobius, which is synonymized here with Apollonia; containing the type species N. fluviatilis, along with N. melanostomus and N. caspius), Ponticolini (containing the genera Mesogobius, Proterorhinus, Babka, and Ponticola—elevating the latter two from subgenera and removing them from the formerly paraphyletic Neogobius), and Benthophilini (tadpole gobies). Within Ponticolini, Proterorhinus and Mesogobius comprise the sister clade of the Ponticola and Babka clade. Further work is needed to clarify the interrelationships of the tadpole gobies. Invasiveness is widespread in freshwater and euryhaline taxa of Neogobius, Proterorhinus, Babka, and Ponticola; but not in marine species, Mesogobius, or tadpole gobies.

Molecular Phylogenetics and Evolution 52 (2009) 84–102 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Escape from the Ponto-Caspian: Evolution and biogeography of an endemic goby species flock (Benthophilinae: Gobiidae: Teleostei) Matthew E. Neilson, Carol A. Stepien * Great Lakes Genetics Laboratory, Lake Erie Center and Department of Environmental Sciences, University of Toledo, 6200 Bayshore Rd., Toledo, OH 43618, USA a r t i c l e i n f o Article history: Received 29 September 2008 Revised 22 December 2008 Accepted 30 December 2008 Available online 14 January 2009 Keywords: Apollonia Babka Gobiidae Mesogobius Mitochondrial gene phylogeny Nuclear gene phylogeny Neogobius Ponticola Ponto-Caspian Proterorhinus a b s t r a c t Endemic Ponto-Caspian gobies include a flock of 24 ‘‘neogobiin” species (containing the nominal genera and subgenera Apollonia, Babka, Neogobius, Mesogobius, Ponticola, and Proterorhinus; Teleostei: Gobiidae), of which a large proportion (5 species; 21%) recently escaped to invade other freshwater Eurasian systems and the North American Great Lakes. We provide its first comprehensive phylogenetic and biogeographic analysis based on 4709 bp sequences from two mitochondrial and two nuclear genes with maximum parsimony, likelihood, and Bayesian approaches. We additionally compare its relationships with the tadpole gobies (Benthophilus and Caspiosoma), which comprise a related endemic Ponto-Caspian gobiid group; along with a variety of postulated relatives and outgroups. Results of all phylogenetic approaches are highly congruent and provide very strong support for recognizing the subfamily Benthophilinae; which encompasses both the ‘‘neogobiins” and tadpole gobies, and genetically diverges from other Gobiidae subfamilies—including (non-monophyletic) Gobiinae and Gobinellinae. Benthophilinae contains three tribes: Neogobiini (Neogobius, which is synonymized here with Apollonia; containing the type species N. fluviatilis, along with N. melanostomus and N. caspius), Ponticolini (containing the genera Mesogobius, Proterorhinus, Babka, and Ponticola—elevating the latter two from subgenera and removing them from the formerly paraphyletic Neogobius), and Benthophilini (tadpole gobies). Within Ponticolini, Proterorhinus and Mesogobius comprise the sister clade of the Ponticola and Babka clade. Further work is needed to clarify the interrelationships of the tadpole gobies. Invasiveness is widespread in freshwater and euryhaline taxa of Neogobius, Proterorhinus, Babka, and Ponticola; but not in marine species, Mesogobius, or tadpole gobies. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Exotic species pose one of the most serious threats to native ecosystems worldwide (Simberloff and Von Holle, 1999; Sax and Gaines, 2008) and often present analytical and conceptual challenges—including resolving their taxonomic identity and systematic relationships. As species introductions increase (Cohen and Carlton, 1998; Lockwood et al., 2006), more nonindigenous taxa will originate from poorly-known groups lacking identification keys and analysis with modern phylogenetic methodology. These problems preclude our understanding of fundamental ecological requirements of introduced taxa, including how they adapt to novel habitats and alter the evolutionary trajectory of native ecosystems (Mooney and Cleland, 2001), thereby impeding effective management or control. Phylogenetic and biogeographic analyses of DNA sequence data, as accomplished here, thus provide us with the means to identify invasive taxa, elucidate cryptic species, ana* Corresponding author. Fax: +1 419 530 8399. E-mail addresses: matthew.neilson@utoledo.edu (M.E. Neilson), carol.stepien@utoledo.edu (C.A. Stepien). 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2008.12.023 lyze whether congeners and relatives invade in concert, and predict potential new invaders. For example, the ecology of the North American Great Lakes recently has been restructured by waves of invaders accidentally introduced from ships’ ballast water, primarily from the Eurasian Ponto-Caspian region (including the Aral, Azov, Black, and Caspian Seas and associated drainages; Mills et al., 1993; Ricciardi and MacIssac, 2000). Notable for their ecological effects are the dreissenid zebra and quagga mussels, Dreissena polymorpha and D. bugensis, which first appeared in the Great Lakes in the mid-1980s via ballast water introduction. Two Ponto-Caspian gobies then entered the Great Lakes in 1990 (Crossman et al., 1992)—the round goby Neogobius melanostomus (Pallas 1814) (= Apollonia melanostoma per Stepien and Tumeo, 2006) and the freshwater tubenose goby Proterorhinus semilunaris (Heckel 1837) (formerly grouped as a single species with the marine P. marmoratus [Pallas 1814]; Stepien and Tumeo, 2006). Like the zebra mussel, the round goby spread rapidly throughout all five Great Lakes (USGS, 2003) and is now one of the most abundant benthic fish species (Jude and DeBoe, 1996; Johnson et al., 2005). Its invasion success likely was aided by the prevalence of its native dreissenid mussel prey (Ray and M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 Corkum, 1997). Such facilitative interactions among co-evolved invaders may significantly augment the success of invasive communities (Simberloff and Von Holle, 1999; Ricciardi and MacIssac, 2000), with widespread ecological consequences—as has occurred with the growing dominance of the dreissenid mussel/round goby benthic community (Vanderploeg et al., 2002). The round and tubenose gobies are members of an enigmatic group native to the Ponto-Caspian region containing 24 species arranged (prior to the present study) in four genera (Apollonia, Mesogobius, Neogobius, and Proterorhinus; Miller, 2003b; Stepien and Tumeo, 2006; see Table 1), which have been variously termed ‘‘neogobiins”. Several taxa also contain putative subspecies divided between the Black and Caspian Sea basins. This group meets the definition of a species flock sensu Greenwood (1984)—a geographically circumscribed, monophyletic taxon characterized by marked radiation. The historic endemism and taxonomic diversity of the Ponto-Caspian ‘‘neogobiins” are remarkable, and knowledge of their evolutionary history may yield insight on the evolution of species flocks (Johns and Avise, 1998), factors leading to their rapid evolutionary diversification, as well as invasive success in new habitats. Despite their remarkable radiation, the systematic relationships and placement of Ponto-Caspian ‘‘neogobiin” gobies have been disputed and unclear. This phylogenetic confusion is highlighted by the fact that a large number of the group are invasive [5 species; 21%; including the round goby N. melanostomus, monkey goby N. fluviatilis (= A. fluviatilis) (Pallas 1814), racer goby N. gymnotrachelus (Kessler 1857), bighead goby N. kessleri (Gunther 1861), and freshwater tubenose goby P. semilunaris] in freshwater systems of Eastern/Central Europe and/or North America. For example, N. melanostomus is invasive in both Europe and North America, and in the latter has undergone rapid range expansion since its 1990 introduction (Charlebois et al., 1997) and is implicated in the decline of native Great Lakes fishes (Jude et al., 1995; Corkum et al., 2004). Detailed investigations of morphology, osteology, and systematics of the Ponto Caspian ‘‘neogobiin” gobies have only recently begun; and relationships of the genus Neogobius sensu lato with other taxa have been disputed. For example, members of the genera Neogobius sensu lato and Proterorhinus were regarded as subgenera of Gobius by Vasil’eva (1989, 1991, 1999) based on cranial osteology; which is a historic perspective predating Berg (1949) that was not accepted by the research community (see Miller, 2003). Birdsong et al. (1988), in a study of vertebral column and median fin osteology, failed to place Neogobius (the sole Ponto-Caspian representative in their study) in any of their hypothesized genus-groups, whereas Pezold (1993) proposed that this genus may belong to the subfamily Gobionellinae based on its patterning of infraorbital pores in the cephalic lateral line system (although he did not directly examine any ‘‘neogobiin” material). Simonovic (1999) described divergence for Neogobius sensu lato and Proterorhinus of ‘‘neogobiins” from other taxa in the subfamily Gobiinae, hypothesizing close relationship but distinctiveness between ‘‘gobiins” and ‘‘neogobiins”, based on external morphometrics, osteology, and karyology, but did not examine Mesogobius. Ahnelt and Duchkowitsch’s (2004) study of postcranial osteology of Proterorhinus placed it along with Neogobius sensu lato in the Gobiinae. Composition of the genus Neogobius sensu lato and its interrelationships also have been controversial. Miller and Vasil’eva (2003) summarized information, listing the genus as comprising 14 species separated into five subgenera—N. Apollonia (containing only N. melanostomus), N. Neogobius (restricted to N. fluviatilis), N. Eichwaldiella (containing only N. caspius), N. Babka (= N. gymnotrachelus), and N. Ponticola (including N. cephalargoides, kessleri, ratan, syrman, etc.). Miller (2003b) elevated the subgenus N. Chasar to generic status (containing a single taxon—C. bathybius) based on 85 increased modal number of dorsal fin rays and differences in the pattern of cephalic sensory papillae; however, this distinction is questionable and the taxon is regarded as belonging in N. Ponticola (E. Vasil’eva, personal communication). Stepien and Tumeo (2006) elevated Apollonia (including N. melanostomus and N. fluviatilis) to generic status due to its paraphyletic position relative to the other subgenera of Neogobius sensu lato (i.e. N. Babka and N. Ponticola), based on mitochondrial DNA cytochrome b gene sequences. Moreover, the clade containing Proterorhinus, Mesogobius, Apollonia sensu Stepien and Tumeo (2006), and Neogobius sensu lato appeared separated from the Gobiinae tested (Gobius and Zosterissessor; Stepien and Tumeo, 2006). Miller and Vasil’eva (2003) noted that the systematic relationships of the Ponto-Caspian gobies are poorly understood, provided no hypothesis for their relationships, and expressed the need for a detailed cladistic revision. Although some prior studies examined selected morphological aspects of their relationships and systematics, none investigated relationships of the native Ponto-Caspian gobies as a whole and only those by our laboratory and one other used a molecular approach (i.e., partial group analyses by Dougherty et al., 1996; Dillon and Stepien, 2001; Stepien and Tumeo, 2006; Neilson and Stepien, 2009). Our central goal is to analyze the systematic relationships among Ponto-Caspian ‘‘neogobiin” gobies, and to illuminate some of the factors (biogeographic, evolutionary, or phylogenetic) leading to their diversification that also may augment their success as invasive species. In particular, we investigate the following questions: (1) are the currently recognized species of ‘‘neogobiin” gobies valid (i.e., reciprocally monophyletic) taxa? (2) are the current genera valid?, and (3) how was their speciation and diversification shaped by the geologic history of the Ponto-Caspian region? We analyze the phylogenetic relationships among ‘‘neogobiin” gobies, in comparison with gobiin relatives and outgroup taxa, using DNA sequence data from four gene regions: the mitochondrial (mt) cytochrome (cyt) b and cytochrome oxidase c subunit I (COI) genes, and the nuclear recombination activating gene 1 (RAG1) and S7 ribosomal protein intron 1 (S7). We include the 19 most prevalent members of 24 nominal species (Miller, 2003b; Freyhof and Naseka, 2007; Kovacic and Engin, 2008; Neilson and Stepien, 2009; see Table 1) in the most complete phylogenetic study of the group. 2. Methods 2.1. Taxon sampling Taxa analyzed in this study, collection locations, and corresponding GenBank accession numbers (http://www.ncbi.nlm.nih.gov) are listed in Table 1. Specimens were collected by us and colleagues throughout the range of the ‘‘neogobiins” within the Ponto-Caspian region (Fig. 1) via small seines, beam/otter trawls, or by hook and line, and include all widely distributed and common taxa. We analyze all proposed subgenera of Neogobius sensu lato (= N. Babka, N. Eichwaldiella, and N. Ponticola; Miller and Vasil’eva, 2003) and the genera Apollonia sensu Stepien and Tumeo (2006), Proterorhinus, and Mesogobius; absent taxa either are very rare (e.g., Mesogobius nonultimus), confined to deeper water (N. bathybius), were only recently described (N. rizensis and N. turani; Kovacic and Engin, 2008), or have extremely limited distributions (Proterorhinus tataricus; Freyhof and Naseka, 2007). We include seven Gobiinae outgroups (Chromogobius zebratus, Gobius auratus, G. bucchichi, G. fallax, G. niger, Pomatoschistus minutus and Zosterisessor ophiocephalus) that range throughout the Black and Mediterranean Seas and are members of the hypothesized sister lineage of the Ponto-Caspian gobiids (Miller, 1990). We also utilize five species of tadpole gobies, including Benthophilus (B. abdurahmanovi, 86 Table 1 Taxonomic names, geographic origin, GenBank accession numbers, specimen ID, and type species for the genus () and tribe () for individuals/taxa analyzed in the present study. Former taxon and author Common name Location Latitude Longitude Specimen ID GenBank Accession Nos. cyt b COI RAG1 S7 Tribe Neogobiini Neogobius = Apollonia Iljin 1927 A. fluviatilis = N. fluviatilis (Pallas 1814)** A. melanostoma = N. melanostomus ?tul?> (Pallas 1814) N. caspius (Eichwald 1831) Monkey Goby Danube River, Vilkove, Ukraine 45.393989 29.586870 AGV7 FJ526749 FJ526804 FJ526858 FJ526913 46.655616 46.016147 48.870870 46.272008 35.278634 43.448435 44.660139 45.615373 AGV9 ANT5 ALL13 ANG11 FJ526750 FJ526753 FJ526751 FJ526752 FJ526805 FJ526808 FJ526806 FJ526807 FJ526859 FJ526862 FJ526860 FJ526861 FJ526914 FJ526917 FJ526915 FJ526916 Round Goby Sea of Azov, Molochnyi, Ukraine Ozero Manych, Prujitnoe, Russia Volga River, Volgograd, Russia Chernozemelskii Canal, near Elista, Russia Dnieper River, Kiev, Ukraine 50.270000 30.300000 AHC3 EU331208 FJ526799 FJ526853 FJ526908 Black Sea, Sevastopol, Ukraine Kerch Strait, Kerch, Ukraine Volga River, Svetli Yar, Russia Caspian Sea, Nabran, Azerbaijan Caspian Sea, Nabran, Azerbaijan Caspian Sea, Sumgait, Azerbaijan 44.604040 45.358334 48.484638 41.837222 41.837222 40.600278 33.540840 36.475834 44.784676 48.620000 48.620000 49.682222 AHF8 APC8 AMP2 AKB1 APT1 ALK6 EU331225 EU331173 EU331175 EU331186 FJ526756 FJ526757 FJ526800 FJ526803 FJ526802 FJ526801 FJ526811 FJ526812 FJ526854 FJ526857 FJ526856 FJ526855 FJ526865 FJ526866 FJ526909 FJ526912 FJ526911 FJ526910 FJ526921 FJ526922 Kanev Reservoir, Kiev, Ukraine 50.270000 30.300000 AJE3 FJ526755 FJ526810 FJ526864 FJ526920 Black Sea, Odessa, Ukraine Sea of Azov, Molochnyi, Ukraine 46.470820 46.655616 30.735090 35.278634 AGV10 AGV11 EU444668 FJ526754 EU444697 FJ526809 EU444723 FJ526863 FJ526918 FJ526919 Pinchuk’s Goby Dniester River Estuary, Ukraine 46.066667 30.450000 ALC12 FJ526758 FJ526813 FJ526867 FJ526923 Kerch Strait, Kerch, Ukraine Dniester River Estuary, Ukraine Khobi River, Khobi, Georgia 45.358334 46.066667 42.317778 36.475834 30.450000 41.916111 AGT10 ATW11 ATW05 FJ526773 FJ526794 FJ526790 FJ526828 FJ526850 FJ526846 FJ526882 FJ526904 FJ526900 FJ526939 FJ526978 FJ526974 Otap River, Otap, Georgia Aragvi River, Tsiteltsopeli, Georgia Ptsa River, Georgia Cape Langeron, Odessa Bay, Ukraine 42.918333 41.993611 42.04972 46.483333 41.541944 44.760278 43.72889 30.755000 ATW06 ATW03 ATW04 ALC7 FJ526791 FJ526788 FJ526789 FJ526759 FJ526847 FJ526844 FJ526845 FJ526814 FJ526901 FJ526898 FJ526899 FJ526868 FJ526975 FJ526972 FJ526973 FJ526924 Sukhyi Estuary, Burlachya Balka, Ukraine Cape Malyi Fontan, Odessa Bay, Ukraine Karpovska Reservoir, Iliovka, Russia. 46.326700 30.667550 ALC8 FJ526760 FJ526815 FJ526869 FJ526925 46.450000 30.766667 ALC9 FJ526761 FJ526816 FJ526870 FJ526926 48.643269 43.617069 AKS4 FJ526762 FJ526817 FJ526871 FJ526927 Caspian Sea, Nabran, Azerbaijan Caspian Sea, Lenkoran, Azerbaijan 41.837222 38.751944 48.620000 48.868889 APT3 APT4 FJ526763 FJ526764 FJ526818 FJ526819 FJ526872 FJ526873 FJ526928 FJ526929 Caspian Goby Tribe Ponticolini Mesogobius Bleeker 1874 Mesogobius batrachocephalus (Pallas 1814)* Neogobius Iljin 1927 N. cephalargoides Pinchuk 1976 Knout Goby Ponticola Iljin 1927 Ponticola cephalargoides (Pinchuk 1976) N. constructor (Nordmann 1840) Po. constructor (Nordmann 1840) Constructor Goby N. cyrius (Kessler 1874) Po. cyrius (Kessler 1874) Kura Goby N. eurycephalus (Kessler 1874) Po. eurycephalus (Kessler 1874) Ginger Goby N. gorlap (Iljin 1949) Po. gorlap (Iljin 1949) Caspian Bighead Goby M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 Proposed name (otherwise taxon to the left is current) N. kessleri (Günther 1861) Po. kessleri (Günther 1861) Bighead Goby Danube River, Dobra, Serbia 44.638100 21.909400 APT8 FJ526770 FJ526825 FJ526879 FJ526936 Dniester River, Yampil, Ukraine Simferopol Reservoir, Simferopol, Ukraine 48.235344 44.921746 28.293024 34.155719 ALC2 APT7 FJ526768 FJ526769 FJ526823 FJ526824 FJ526877 FJ526878 FJ526934 FJ526935 Po. platyrostris (Pallas 1814) Flatsnout Goby Kerch Strait, Kerch, Ukraine 45.358334 36.475834 AGT7 FJ526771 FJ526826 FJ526880 FJ526937 N. ratan (Nordmann 1840) Po. ratan (Nordmann 1840)** Ratan Goby Kerch Strait, Kerch, Ukraine Sea of Azov, Ukraine 45.358334 45.782058 36.475834 35.487513 AGT9 ATW07 FJ526772 FJ526792 FJ526827 FJ526848 FJ526881 FJ526902 FJ526938 FJ526976 N. rhodioni Vasil’eva and Vasil’ev 1994 Po. rhodioni (Vasil’eva and Vasil’ev 1994) Rhodion’s Goby 45.782058 43.70333 35.487513 39.68889 ATW08 ATW01 FJ526793 FJ526786 FJ526849 FJ526842 FJ526903 FJ526896 FJ526977 FJ526970 N. syrman (Nordmann 1840) Po. syrman (Nordmann 1840) Syrman Goby Sea of Azov, Ukraine Vostochnyy Dagomys River, Baranovka, Russia Kherota River, Moldovka, Russia Danube River, Vilkove, Ukraine 43.46444 45.393989 39.95333 29.586870 ATW02 AGV4 FJ526787 FJ526774 FJ526843 FJ526829 FJ526897 FJ526883 FJ526971 FJ526940 Danube River, Vilkove, Ukraine 45.393989 29.586870 AJE8 FJ526775 FJ526830 FJ526884 FJ526941 Neogobius Iljin 1927 Neogobius gymnotrachelus (Kessler 1857) Babka Iljin 1927 Babka gymnotrachelus (Kessler 1857)* Dniester River delta, Bilyayivka, Ukraine 46.468333 30.216667 AMU6 FJ526765 FJ526820 FJ526874 FJ526930 Dnieper River, Kiev, Ukraine Kanev Reservoir, Kiev, Ukraine Tyligul Estuary, Ukraine 50.270000 50.270000 46.470820 30.300000 30.300000 30.735090 AGT1 AGT3 AGT2 FJ526766 EU444667 FJ526767 FJ526821 EU444694 FJ526822 FJ526875 EU444720 FJ526876 FJ526931 FJ526932 FJ526933 Dniester River delta, Bilyayivka, Ukraine 46.468333 30.216667 AME1 EU444621 EU444682 EU444708 FJ526942 Cape Langeron, Odessa Bay, Ukraine Tyligul Estuary, Ukraine Black Sea, Sevastopol, Ukraine 46.483333 46.690000 44.604040 30.755000 31.486783 33.540840 AMM1 AMG1 AMR1 EU444624 EU444621 EU444621 EU444687 EU444684 EU444689 EU444713 EU444710 EU444715 FJ526944 FJ526943 FJ526945 Lake Superior, MI, USA 46.666667 92.200000 AOC2 EU444607 EU444690 EU444716 FJ526948 Lake St. Clair, Michigan, USA Danube River, Dobra, Serbia Dniester River, Mohyliv-Podil’sky, Ukraine Kurchurgan Reservoir, Hradenytsi, Ukraine Cape Malyi Fontan, Odessa Bay, Ukraine Simferopol Reservoir, Simferopol, Ukraine Karpovska Reservoir, Iliovka, Russia. 42.594282 44.638100 48.449428 46.100000 46.450000 44.921746 48.643269 82.803323 21.909400 27.778285 30.200000 30.766667 34.155719 43.617069 AGN1 AKP7 AFE10 AML1 AMF2 AQE1 AKP1 EU444607 EU444612 EU444604 EU444632 EU444626 EU444650 EU444610 EU444674 EU444677 EU444673 EU444686 EU444683 EU444691 EU444675 EU444700 EU444703 EU444699 EU444712 EU444709 EU444717 EU444701 FJ526949 FJ526951 FJ526946 FJ526950 FJ526947 FJ526952 FJ526955 Chagraiskoye Reservoir, Zunda Tolga, Russia Volga River, Preshib, Russia Volga River delta, Russia Volga River, Volgograd, Russia Chernozemelskii Canal, Elista, Russia 45.617691 47.683923 45.788350 48.870870 46.272008 44.211077 46.509057 47.886953 44.660139 45.615373 AMK1 ALT1 AKP4 ALU1 AMN1 EU444630 EU444610 EU444611 EU444611 EU444636 EU444685 EU444679 EU444676 EU444680 EU444688 EU444711 EU444705 EU444702 EU444706 EU444714 FJ526954 FJ526957 FJ526956 FJ526958 FJ526953 Proterorhinus Smitt 1900 Proterorhinus marmoratus (Pallas 1814)* Pr. semilunaris (Heckel 1837) Pr. cf semipellucidus Neilson and Stepien, 2009 Proterorhinus sp. Neilson and Stepien, 2009 Racer Goby Marine Tubenose Goby Freshwater Tubenose Goby Volga Tubenose Goby M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 N. platyrostris (Pallas 1814) 87 88 M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 Fig. 1. Current range (excluding introduced range in North America) of nominal species of the subfamily Benthophilinae (hatched area; based on Miller, 2003), and locations of taxa sampled in the present study. K-M = Kumo-Manych Depression. B. granulosus, B. mahmudbejovi, and B. stellatus) and Caspiosoma caspium. The tadpole gobies constitute a second Ponto-Caspian endemic goby group that is hypothesized to be closely related to ‘‘neogobiins” and their relatives (Ahnelt, 2003). Specimens were preserved immediately following capture either in 95% ethanol for molecular analyses or in 10% formalin (with removal of right pectoral fin for preservation in 95% ethanol and molecular analyses) for future morphological analyses. 2.2. DNA analysis Genomic DNA was isolated from fin clips or caudal muscle tissue using a Qiagen DNEasy tissue kit (Valencia, CA) following manufacturer’s protocols. Two mitochondrial genes (cyt b and COI) and two nuclear genes (RAG1 and S7) were amplified via the polymerase chain reaction (PCR) using the following primers: cyt b—AJG15 (Akihito et al., 2000), H15343goby (Neilson and Stepien, 2009), L15162goby (Neilson and Stepien, 2009), and H5 (Akihito et al., 2000); COI—L6486, H7127, L7057, and H7696 (Thacker, 2003); RAG1—RAG1F1 (López et al., 2004), RAG1-R811goby (Neilson and Stepien, 2009), RAG1-F709 (50 -CTTATGTCTGCACGCTCTGC-30 , this study), and RAG1R1 (López et al., 2004); and S7—S7RPEX1F and S7RPEX2R (Chow and Hazama, 1998). PCR amplifications were performed in 25 lL volumes containing 10 mM Tris–HCl pH 8.3, 50 mM KCl, 1.5 mM MgCl2 (2.5 mM for COI), 0.001% (w/v) gelatin, 200 lM each dNTP, 0.5 lM each primer, 1.5 units of Taq polymerase, and 100 ng (1–3 lL) of template DNA. The PCR profile for cyt b and RAG1 included an initial denaturation of 94 °C for 2 min, 40 cycles of 94 °C for 45 s, gene specific annealing temperature (cyt b—52°; RAG1—50°; S7—60°) for 30 s, and 72 °C for 60 s, with a final extension of 72 °C for 3 min. The cycling profile for COI included an initial denaturation of 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 60 s, with a final extension of 72 °C for 2 min. The profile for S7 included an initial denaturation of 94 °C for 3 min, 40 cycles of 94 °C for 60 s, 60 °C for 45 s, and 72 °C for 120 s, with a final extension of 72 °C for 5 min. PCR reactions were checked on 1% agarose gels stained with ethidium bromide, and excess primers and unincorporated nucleotides were removed from successful reactions with spin column purification kits (QIAquick PCR Purification Kit, Qiagen; or QuickStep 2 PCR Purification Kit, Edge Biosystems, Gaithersburg, MD). For all genes, amplicons were sequenced in both directions using dye-labeled terminators and PCR primers, and resolved on an ABI 3730 (Applied Biosystems, Foster City, CA) genetic analyzer at the Cornell University Life Sciences Core Laboratories Center. Forward and reverse sequences for each gene per individual were aligned in our laboratory, a contiguous sequence was created with BioEdit (Hall, 1999), and sequences for each gene were globally aligned using Clustal X (cyt b, COI, RAG1; Larkin et al., 2007) or T-Coffee (S7; Notredame et al., 2000). 2.3. Phylogenetic analyses We used parsimony, likelihood, and Bayesian approaches to reconstruct phylogenies, with PAUP* v4.0b10 (Swofford, 2003), PhyML v2.4.4 (Guindon and Gascuel, 2003) and MrBayes v3.1 (Ronquist and Huelsenbeck, 2003), respectively. Parsimony analyses were performed using unweighted heuristic searches, with starting trees obtained by random addition (100 replications) holding 10 trees per replicate, and tree-bisection-reconnection branch swapping. Branch support was calculated for the inferred branches via non-parametric bootstrapping (2000 replications) (Table 2). For likelihood and Bayesian analyses, ModelTest v3.7 (Posada and Crandall, 1998) was employed to determine the simplest best-fit model of evolution for each gene under the Akaike information criterion. For the cyt b data, the best-fit model was M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 Table 2 Summary of maximum parsimony results from individual genes and from combined dataset using PAUP* v4b10 (Swofford, 2003). Gene N trees Length CI RI RC HI COI cyt b RAG 1 S7 Combined 6 64 533503 6.3 106 54 1541 1639 215 367 3879 0.447 0.475 0.758 0.730 0.490 0.868 0.882 0.950 0.902 0.875 0.388 0.419 0.720 0.659 0.428 0.553 0.525 0.242 0.270 0.510 CI—consistency index; RI—retention index; RC—rescaled consistency index; HI— homoplasy index. GTR + I + G with a shape parameter (a) = 0.9552 and proportion of invariant sites (i) = 0.4627. For the COI gene, the GTR + I + G also was selected (a = 1.1661; i = 0.5770). For the nuclear genes, the best-fit model was TrN + G for RAG1 (a = 0.6191) and TVM + G (a = 1.1150) for S7. Bayesian analyses using a Metropolis coupled Markov chain Monte Carlo (MCMCMC) approach were run for 5 million generations, with sampling every 100 generations, to ensure convergence of likelihood values. Four separate chains were run simultaneously for each analysis, and two analyses were run simultaneously. The burn-in period for the MCMCMC analysis was determined by plotting log likelihood values at each generation to identify the point at which they reached stationarity. In all analyses, stationarity was reached by 50,000 generations; thus, a conservative burn-in period of 500,000 generations was used, and trees and parameter values sampled prior to the burn-in were discarded. Branch support for likelihood analyses was calculated using non-parametric bootstrapping (2000 replications) and via the posterior probability distribution of clades for Bayesian analyses. In addition to the separate analyses, we explored the combinability of the four gene regions into a single dataset using several methods. An incongruence length difference (ILD) test (Farris et al., 1995) in PAUP* (1000 replications) was employed to determine the congruence of topologies among datasets within a parsimony framework, using heuristic searches with 50 random addition sequences per replicate. As the ILD test is known to be susceptible to noise within datasets, all uninformative characters were removed. Significant incongruence was found among all four genes, as well as within and between mitochondrial and nuclear genomes (p = 0.001 in all tests). To further explore the extent and location of congruence, we calculated partitioned branch support (a.k.a. partitioned Bremer support [PBS]; Baker and DeSalle, 1997) for each gene region, and the partition congruence index (PCI; Brower, 2006) for the four genes combined. Briefly, partitioned branch support determines the contribution of each partition to the total branch support for each branch on a phylogeny, with the sum of PBS for all partitions equaling the total branch support (BS) for each individual branch. The partition congruence index (PCI) incorporates the magnitude of difference between PBS for each partition and the total BS of all partitions combined, and thus summarizes the amount of incongruence among the partitions (Brower, 2006). When all partitions are congruent, PCI is equal to the BS of an individual branch; as the amount of incongruence among partitions increases, PCI decreases linearly and eventually becomes negative at high levels of incongruence. As conflict among the four analyzed genes (i.e., negative values of PBS for one or more genes; PCI values <0 or substantially less than BS) was limited primarily to intraspecific branches and our deeper interspecific and intergeneric branches were strongly supported, we conducted a second series of phylogenetic analyses using the combined four gene dataset. Search strategies for the concatenated sequences were identical to those in the separate analyses. ModelTest selected the 89 TVM + I + G model (a = 0.4680; i = 0.3331) as the best fit model for the combined sequence data. A partitioned mixed-model approach was used for Bayesian analysis of the combined gene regions. Models of sequence evolution identified for each individual gene region were assigned using the APPLYTO command, and the appropriate model parameters were estimated for each gene using the UNLINK command. Topological differences among the three different analysis methods for the concatenated sequences were tested with a Shimodaira and Hasegawa (1999) test (10000 RELL bootstrap replicates) implemented in PAUP*. To examine the placement of the Ponto-Caspian ‘‘neogobiin” gobies among other gobioid fishes, we performed additional phylogenetic analyses. The first was performed using cyt b sequences from the present study as well as additional taxa collected by us and sequences from GenBank, including: Gobiidae: Amblyopinae—Taenioides limicola Smith 1964 (AB021253); Gobiinae—Elacatinus macrodon (Beebe & Tee-Van 1928) (AY846447), Gobiosoma bosc (Lacepède 1800) (AY848456), Knipowitschia caucasica (Berg 1916) (FJ526796); Gobionellinae—Acanthogobius flavimanus (Temminck & Schlegel 1845) (AB021249), Gymnogobius petschiliensis (Rendahl 1924) (AY525784), Rhinogobius giurinus (Rutter 1897) (AB018997), Tridentiger bifasciatus Steindachner 1881 (AB021254); Oxudercinae—Periophthalmus argentilineatus Valenciennes 1837 (AB021251); Eleotridae: Butinae—Butis amboinensis (Bleeker 1853) (AB021232), Ophiocara porocephala (Valenciennes 1837) (AB021245); Eleotrinae—Dormitator maculatus (Bloch 1792) (AB021234), Eleotris fusca (Forster 1801) (AB021236); Kraemeriidae—Kraemeria cunicularia Rofen 1958 (AB021250); Microdesmidae—Gunnellichthys monostigma Smith 1958 (AB021256); Odontobutidae—Odontobutis obscura (Temminck & Schlegel 1845) (AB021243), Odontobutis platycephala Iwata & Jeon 1985 (DQ010651); Ptereleotridae—Ptereleotris heteroptera (Bleeker 1855) (AB021252); Rhyacichthyidae—Rhyacichthys aspro (Valenciennes 1837) (AP004454). The second analysis was run using COI sequence data from the present study combined with COI sequences obtained by Thacker (2003) in an analysis of the molecular systematics of gobioid fishes. Phylogenetic analyses for the expanded cyt b and COI datasets were conducted as described above, with the GTR + I + G model (a = 0.600; i = 0.423) chosen as the best-fit model for cyt b, and also as the best-fit model for COI (a = 0.589; i = 0.547). 2.4. Divergence time estimation To estimate divergence times among major lineages, we used a penalized likelihood approach (Sanderson, 2002) implemented in the program r8s 1.71 (Sanderson, 2003). An initial age estimate was generated for the extended cyt b ML tree under a molecular clock assumption, from which our sequences significantly departed, and a second analysis was conducted using penalized likelihood with the optimal smoothing parameter (= 3.2) determined by cross-validation in r8s. Divergence time estimates under penalized likelihood require a fixed age for at least one node within the phylogeny. Rückert-Ülkümen (2006) described fossil otoliths of Neogobius as dating to the late Miocene-early Pliocene (10 Mya), and Bajpai and Kapur (2004) describe the earliest fossils of Gobiidae from the early Eocene (51–56 Mya). Thus, we followed Neilson and Stepien (2009) and set the age of the node for Gobiidae to 53 My and the most recent common ‘‘neogobiin” ancestor to 10 My, as their otoliths are morphologically similar. 3. Results The dataset for the combined four gene regions across 19 ‘‘neogobiin” taxa and 12 outgroups comprises 4709 aligned bp (cyt b— 1142 bp; COI—1271 bp; RAG1—1556 bp; S7—740 bp including in- 90 M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 dels). GenBank accession numbers are EU331173, EU331175, EU331186, EU331208, EU331225, EU444604, EU444607, EU444610–EU444612, EU444621, EU444624, EU444626, EU444630, EU444632, EU444636, EU444650, EU444667, EU444668, EU444670, and FJ526747–FJ526795 for cyt b; EU444673–EU444677, EU444679–EU444680, EU444682– EU444691, EU444694, EU444697–EU444698, and FJ526797– FJ526850 for COI; EU444699–EU444700, EU444702–EU444703, EU444705–EU444706, EU444708–EU444717, EU444720, EU444723–EU444724, and FJ526851–FJ526904 for RAG1; and FJ526905–FJ526978 for S7 (Table 1, Appendix A; 61 individuals). Base composition is stationary across taxa for each gene (v2 > 31.6; df = 180; p > 0.29). Phylogenies inferred from the parsimony (MP), likelihood (ML), and Bayesian (BI) analyses of the combined four gene dataset generally are highly congruent: no statistical significant differences are found among the three topologies (S-H test, p = 0.21), and the ML tree is presented in Fig. 2 for clarity. Our results reveal a mono- Fig. 2. Maximum likelihood phylogeny (PhyML; Guindon and Gascuel, 2003) of the subfamily Benthophilinae and outgroups based on combined analysis of four gene regions. Numbers at nodes indicate likelihood bootstrap support (2000 pseudoreplications), with * = 100%. M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 phyletic clade containing all Ponto-Caspian endemic gobiid taxa— redefined and redescribed in the present study as Benthophilinae Beling and Iljin 1927—which is the oldest historic subfamilial name. The subfamily Benthophilinae is clearly divergent and distinct from other gobiid taxa, and contains both the former ‘‘neogobiins” as well as the tadpole gobies. All genera and all individual nominal species are highly supported (>95% bootstrap support for MP and ML analyses, >4 branch support for MP, and >0.95 posterior probability for BI). Three clades are highly resolved in Benthophilinae, showing that the genus Neogobius sensu lato (containing the subgenera/genera Apollonia sensu Stepien and Tumeo [2006], Neogobius, Babka, and Ponticola; see Table 1) is paraphyletic and invalid. Notably, Apollonia sensu Stepien and Tumeo (2006) and the former Neogobius sensu lato are each separated by the genera Proterorhinus and Mesogobius (Fig. 2). The three primary clades of Benthophilinae (corresponding to tribes within the subfamily) are (1) a now-restricted Neogobius (= Apollonia, containing the type species N. fluviatilis) that is monotypic in the tribe Neogobiini, (2) the tadpole gobies, including the genera Benthophilus and Caspiosoma, comprising the tribe Benthophilini, and (3) a larger clade termed the Ponticolini, which contains Proterorhinus, Mesogobius, and Iljin’s (1927) former subgenera Babka and Ponticola that we here elevate to the level of genera (removing them from Neogobius sensu lato). Our results demonstrate high support for a now-restricted genus Neogobius (= Apollonia per Stepien and Tumeo, 2006), which contains N. fluviatilis (= A. fluviatilis) + N. melanostomus (= A. mela- 91 nostoma) + N. caspius (>83% bootstrap support, 12 branch support, 1.00 posterior probability). Our trees show that the former subgenera Babka (containing B. gymnotrachelus) and Ponticola (comprising Po. cephalargoides, Po. constructor, Po. cyrius, Po. eurycephalus, Po. gorlap, Po. kessleri, Po. platyrostris, Po. ratan, Po. rhodioni, and Po. syrman) are each strongly supported as separate clades (>97% bootstrap support, >14 branch support, 1.00 posterior probability), clearly diverge from the other genera, and warrant elevation to generic status. The genera Mesogobius and Proterorhinus are strongly supported as sister groups (>87% bootstrap, 9 branch support, 1.00 posterior probability), and a Mesogobius + Proterorhinus clade is then the sister clade to Babka and Ponticola (100% bootstrap support, 63 branch support, 1.00 posterior probability). The two tadpole goby genera Benthophilus and Caspiosoma comprise the tribe Benthophilini, which constitutes the sister clade to the new tribe Ponticolini (Babka, Ponticola, Mesogobius, and Proterorhinus). Level of support for this sister relationship varies among analysis methods (high parsimony branch support and posterior probability, lower parsimony bootstrap support, very low likelihood bootstrap support) and represents a short internal branch on our phylogeny. The now-restricted Neogobius (= Apollonia) is strongly supported (>95% bootstrap, 37 branch support, 1.00 posterior probability) as the sister clade of the Ponticolini + Benthophilini. This result confirms the relationship described by Stepien and Tumeo (2006) and Neilson and Stepien (2009), of a restricted Neogobius (= Apollonia) as a separate genus from Ponticola and Babka. Fig. 3. Chronogram for the subfamily Benthophilinae and related Ponto-Caspian gobies, derived from a penalized likelihood analysis of divergence time (r8s; Sanderson, 2003) and maximum likelihood analysis of the extended cyt b dataset. Nodes with fixed ages in divergence time analysis are lettered; numbers at nodes indicate support values from phylogenetic analyses of the extended cyt b dataset (likelihood bootstrap). 92 M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 Results of extended cyt b analyses are very similar to the combined four gene analyses (ML topology and support values in Fig. 3), with slight placement differences for Ponticola ratan and Po. syrman, and in grouping the Benthophilini (Benthophilus + Caspiosoma) with the Neogobiini rather than with the Ponticolini. There is strong support for the subfamily Benthophilinae as a clade distinct from the remainder of the Gobiidae, as well as for generic separation of Neogobius (= Apollonia), Babka, and Ponticola. Divergence time estimates from the extended cyt b ML tree are reported in Table 3. Separation of the subfamily Benthophilinae from other Gobiinae taxa occurred 39 million years ago (Mya). The genera Neogobius, Mesogobius, Proterorhinus, Babka, Ponticola, Benthophilus, and Caspiosoma) have similar dates of origin, ranging from 4.29–6.25 Mya. Proterorhinus diverged from Mesogobius 6.18 Mya. Among the remaining genera, the three Neogobius (= Apollonia) species are separated by an estimated 5.47 My, Proterorhinus species by 4.29 My, and Babka and Ponticola by 5.08 My. Within Ponticola, a split (4.07 My) into two primary clades occurred: a ‘‘kessleri” group containing Po. eurycephalus, Po. gorlap, and Po. kessleri, versus a clade containing the remaining Ponticola species. The ‘‘kessleri” group began diverging from one another 1.37 Mya. Within the second Ponticola clade, Po. ratan and Po. syrman branched off soon after separating from the kessleri group; and the remaining species (Po. cephalargoides, Po. constructor, Po. cyrius, Po. platyrostris, and Po. rhodioni) comprise a second ‘‘platyrostris” group that radiated from one another 1.82 Mya. Extended COI analysis trees (Fig. 4) are very similar to the combined four gene analyses, and also are generally congruent with the extended cyt b trees; with all identifying Benthophilinae as a distinct subfamily from other gobiin taxa. The extended COI and extended cyt b analyses vary in degree of separation between the Benthophilinae and members of the putative subfamily Gobiinae, as well as in designating its sister taxa. The extended COI analysis depicts a clade containing Gobius + Zosterisessor as the sister clade of Benthophilinae, which are more distantly related in the extended cyt b analysis. Both extended analyses have similar support values; including high support for the subfamily Benthophilinae and its component taxa, and low support for most deeper gobiid branches outside of the Benthophilinae (Figs. 3 and 4). Phylogenies inferred from the parsimony (MP), likelihood (ML), and Bayesian (BI) analyses primarily differ only in the branching order of individual specimens within species. A single exception for high congruent support in our trees occurs in the freshwater tubenose goby Proterorhinus semilunaris. The evolutionary and phylogeographic history of Proterorhinus recently evaluated by us (Neilson and Stepien, 2009) is very similar to the relationships seen in the present study except for placement of a single individual, Proterorhinus sp. AMN1. In Neilson and Stepien (2009) and the ML and BI analyses here, AMN1 (collected in the Kumo-Manych Depression—a lowland between the Russian Plain and the northern foothills of the Caucasus Mountains; see Fig. 1) groups closely with Proterorhinus from the Caspian Sea/ Volga River clade (Pr. cf semipellucidus); whereas in our current MP analysis, it clusters with the Black Sea freshwater species (Pr. semilunaris). In addition, Pr. semilunaris and Pr. cf semipellucidus are not distinguished as clades in our present MP analysis (Fig. 2); and instead form a single large clade with Pr. semilunaris located basally. This difference likely results from the addition of nuclear S7 intron data, which were not previously sequenced (Neilson and Stepien, 2009). 4. Discussion Phylogenetic analysis of our molecular data for 19 Ponto-Caspian ‘‘neogobiin” species yields a robust phylogeny that generally agrees with prior molecular and morphological data, yet deviates from some earlier morphological hypotheses. Our results reveal that the ‘‘neogobiin” and tadpole ‘‘benthophilin” gobies together comprise a clade that markedly diverges from other gobiid taxa. We thus resurrect and redescribe the subfamily Benthophilinae Iljin 1927 to encompass three tribes; Neogobiini (Neogobius = Apollonia), Ponticolini (containing Babka, Mesogobius, Ponticola, and Proterorhinus), and Benthophilini (the tadpole gobies Benthophilus, Caspisoma, etc.) Our results support the primary findings of Stepien and Tumeo (2006; findings #1–3) and Neilson and Stepien (2009; #2–3) Beling and, which (1) distinguish a restricted genus Neogobius (= Apollonia) comprising the monotypic tribe Neogobiini that is differentiated from the remainder of the Benthophilinae (justify- Table 3 Divergence times for major lineages/nodes within phylogeny of the subfamily Benthophilinae, showing ages estimated for the extended cyt b tree (Fig. 3) using penalized likelihood in r8s (Sanderson, 2003). Nodes representing fixed ages (A and B) and major geologic events in the Ponto-Caspian basin (I–III) are indicated on Fig. 3. Node Estimated age (Mya) MRCA of Gobiidae (A; fixed at 53.00) MRCA of Neogobius sensu stricto, Babka + Ponticola, and Proterorhinus (B; fixed at 10.00) Tribe Neogobiini (Neogobius) + Tribe Benthophilini ‘‘tadpole gobies” (Benthophilus + Caspiosoma) Tribe Ponticolini (Babka + Ponticola + Mesogobius + Proterorhinus) 53.00 10.00 Major geologic event in Ponto-Caspian basin (Reid and Orlova, 2002) Separation of Ponto-Caspian and Pannonian basins (12.5–10 Mya; I) 9.18 7.58 Proterorhinus + Mesogobius 6.25 Tribe Neogobiini = Neogobius sensu stricto (N. fluviatilis, N. melanostomus, and N. caspius) 5.47 Babka (B. gymnotrachelus) + Ponticola (Po. cepharlargoides, Po. constructor, Po. cyrius, Po. eurycephalus, Po. gorlap, Po. kessleri, Po. platyrostris, Po. ratan, Po. rhodioni, and Po. syrman) Tribe Benthophilini ‘‘tadpole gobies” (Benthophilus + Caspiosoma) marine and freshwater tubenose gobies Proterorhinus Ponticola Benthophilus Ponticola ‘‘platyrostris group” (Po. cephalargoides, Po. constructor, Po. cyrius, Po. platyrostris, and Po. rhodioni) Ponticola ‘‘kessleri group” (Po. eurycephalus, Po. gorlap, and Po. kessleri) freshwater Proterorhinus 5.08 5.04 4.29 4.07 2.17 1.82 1.37 1.18 Intermittent connections with World Ocean, with introgression of marine fauna (8.3–6.4 Mya) Brief reconnection with Pannonian basin and immigration of endemic Pannonian fauna (6.4–5.8 Mya) Separation of Black and Caspian basins (5.8–5.0 Mya—coincides with Messinian salinity crisis in paleo-Mediterranean and Black Sea basins; II) Single, large lake in southern Caspian basin (5.2–2.5 Mya, II) Black and Caspian basins connected via Kumo-Manych depression, faunal exchange between basins; glacially-driven fluctuations in water levels (2.6–0.7 Mya; III) M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 93 Fig. 4. Maximum likelihood phylogeny of the goby subfamily Benthophilinae and other gobioids based on COI sequence data from the present study and from Thacker (2003). Numbers above branches indicate likelihood bootstrap support. In clades spanning multiple families/subfamilies, symbols adjacent to species names indicate familial/ subfamilial membership. Pertinent clade described in Thacker (2003) is labeled (IIB). ing its elevation from subgenus to generic status and recognizing it as a divergent tribe), (2) recognize separate marine and freshwater Proterorhinus species in the Black and Caspian Sea basins, and (3) resolve a sister relationship between the genera Proterorhinus and Mesogobius. 4.1. Taxonomic congruency, departures, and nomenclatural changes Our phylogeny differs from some of the prior morphological hypotheses proposed for Benthophilinae relationships. Firstly, we find that Berg’s (1949) grouping of Iljin’s (1927) subgenera of Neo- 94 M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 gobius (Neogobius, Apollonia, Babka, and Ponticola) into a single genus is paraphyletic and invalid. Our trees demonstrate clear phylogenetic separation of a restricted genus Neogobius (= Apollonia) in the tribe Neogobiini from the tribe Ponticolini; which includes the newly elevated genera Babka and Ponticola, along with the genera Proterorhinus and Mesogobius. Berg (1949), as first reviser, selected Neogobius sensu lato as the generic name, and thereby N. fluviatilis became the type species for the genus Neogobius sensu lato. Since the genus name Neogobius must remain as priority for the clade containing N. fluviatilis (W. Eschmeyer, personal communication), we hereby synonymize the generic name Apollonia with Neogobius. In addition, we resolve Neogobius caspius (Eichwald 1831) as belonging to the now-restricted genus Neogobius (N. fluviatilis and N. melanostomus; see Stepien and Tumeo, 2006), which comprises a strongly-supported clade (Figs. 2–4). Neogobius caspius once was placed in a separate (monotypic) subgenus Eichwaldiella (Whitley 1930), and later incorrectly was moved (W. Eschmeyer, personal communication) without justification to a monotypic subgenus Neogobius by Miller and Vasil’eva (2003). Its position relative to other Neogobius/Apollonia species thus was in question prior to our study. Pinchuk (1991) suggested that N. caspius grouped together with N. fluviatilis + N. melanostomus on the basis of mouth size (small in the three species vs. large for taxa now contained in Ponticola and Babka) as well as tooth size distribution on the dentary, but regarded N. caspius as distinct in the forward position of its anterior and posterior nostrils. Miller and Vasil’eva (2003), in describing Iljin’s (1927) subgenera, presented the diagnostic character of an absent metapterygoid/quadrate bridge as uniting N. fluviatilis and N. melanostomus. We discern that the metapterygoid/quadrate bridge likewise is absent in N. caspius, and thus is synapomorphic for our restricted Neogobius clade. Strong support of our molecular data for this restricted Neogobius (= Apollonia) clade (N. caspius + N. fluviatilis + N. melanostomus), combined with several morphological similarities and the nomenclatural changes described above, leads to our redefinition of a restricted Neogobius (in synonymy with Apollonia) in the tribe Neogobiini. The molecular phylogenies presented here, as well as in Stepien and Tumeo (2006) and Neilson and Stepien (2009), are congruent in identifying large separation between Ponticola/Babka and Neogobius sensu stricto/Apollonia. In addition, pronounced genetic divergence between Babka and Ponticola (subgenera delineated by Iljin [1927]), along with their morphological separation and autapomorphies (Miller and Vasil’eva 2003), supports their elevation to generic level. Our phylogenetic trees reflect this new nomenclature. Our results show that the monotypic Babka contains the racer goby B. gymnotrachelus and is the sister species to a strongly-supported monophyletic Ponticola clade, which diverged 4.51– 4.86 Mya (Table 3). The Ponticola + Babka clade is the sister group of Mesogobius + Proterorhinus, with high support; which together form the tribe Ponticolini. Historically, Babka once was hypothesized to be closely related to the knout goby Mesogobius batrachocephalus (Pallas, 1814) based on early studies of morphology (Berg, 1949) and protein electrophoresis (Dobrovolov et al., 1995), although Vasil’ev and Grigoryan (1992) concluded that the two were not congeners based on chromosomal morphology; which is further confirmed by their generic separation in our study. Departures of our phylogeny from former systematic hypotheses occur for the clade Ponticola. Notably, Vasil’eva et al. (1993) suggested two distinct groups within Ponticola based on cranial morphometry: one containing Po. gorlap (Iljin 1949), Po. kessleri, Po. ratan, and Po. syrman (Nordmann 1840), and the other encompassing Po. cephalargoides, Po. eurycephalus (Kessler 1874), Po. platyrostris, and the Caucasian freshwater gobies [Po. constructor (Nordmann 1840), Po. cyrius (Kessler 1874), and Po. rhodioni (Vasil’eva and Vasil’ev 1994)]. Based on our molecular data, Po. ratan is basal to all other Ponticola species; whose branching order differs slightly from that suggested by Vasil’eva et al. (1993). In addition, we resolve two species groups different than those proposed by Vasil’eva et al. (1993): the first group contains Po. eurycephalus, Po. gorlap, and Po. kessleri (designated as the ‘‘kessleri” group in Table 3); the second comprises Po. cephalargoides, Po. constructor, Po. cyrius, Po. platyrostris, and Po. rhodioni (the ‘‘platyrostris” group in Table 3). 4.2. Relationships among Ponto-Caspian endemic gobiid groups Inclusion of the tadpole gobies Benthophilus and Caspiosoma is a novel feature of our molecular phylogeny. Although some recent studies have considered their osteology and taxonomy (e.g., Ahnelt et al., 2000; Ahnelt, 2003; Boldyrev and Bogutskaya, 2007), none of the recent larger-scale studies of goby morphological (Birdsong et al., 1988; Pezold, 1993) or molecular (Akihito et al., 2000; Thacker, 2003) systematics included any tadpole gobies (e.g., Anatirostrum, Benthophiloides, Benthophilus, and Caspiosoma) or ‘‘neogobiin” taxa (Babka, Mesogobius, Neogobius, Ponticola, and Proterorhinus). Although the ‘‘neogobiins” and tadpole gobies were posited to be sister groups based on shared geography and similar postcranial osteology (Ahnelt, 2003), ours is the first study to incorporate both in a comprehensive phylogenetic analysis. All of our analyses strongly support a monophyletic clade comprising the ‘‘neogobiins” and tadpole gobies (Figs. 2–4), for which we resurrect the historic name Benthophilinae (Beling and Iljin 1927), as a subfamily of Gobiidae. Subfamily Benthophilinae contains three distinctive and divergent clades—designated here as the tribes Benthophilini (the tadpole gobies), Neogobiini (monotypic for the genus Neogobius), and Ponticolini (Babka, Mesogobius, Ponticola, and Proterorhinus). Placement of the tribe Benthophilini is inconsistent among some of our trees. All combined sequence data analyses resolve Benthophilini as the sister clade to Ponticolini, however this relationship has mixed support (1.00 posterior probability, <84% bootstrap support, 13 branch support) and relatively short branch lengths. In the extended cyt b analysis Benthophilini is found as the sister clade to Neogobiini (but with short branch length and no support), whereas in the extended COI analysis Benthophilini again groups with Ponticolini. Additional genetic, morphological, and taxonomic sampling of Benthophilini is recommended to further resolve its relationships. One potential indication of a close relationship between Neogobiini and Benthophilini is their shared loss of the metapterygoid bridge, in contrast to its presence in the Ponticolini. However, presence of the metapterygoid bridge is widely considered a pleisiomorphic trait within gobiids (Miller, 1973). 4.3. Higher taxonomic placement of the subfamily Benthophilinae Relationship of the newly-defined Benthophilinae to other gobiids was contentious prior to our study. Morphological studies placed members of the group either in the Gobiinae (Ahnelt, 2003), divergent from other Gobiinae taxa but related to them (Simonovic, 1999), or in the Gobionellinae (Pezold, 1993). Our extended cyt b and COI analyses further resolve this question. In both analyses, the Benthophilinae comprises a true taxon, removed from all other gobiin taxa. In addition, both the cyt b and COI analyses suggest a non-monophyletic Gobiinae similar to that found by Thacker (2003) using the mt COI, ND1, and ND2 genes (who did not examine any ‘‘neogobiins”). Analysis of the COI data from our study combined with Thacker’s (2003) COI data (Fig. 4) yields a similar result to our extended cyt b dataset, depicting a clade comprising the Benthophilinae plus Gobius + Zosterisessor (Gobiinae) nesting as the sister group of one of Thacker’s (2003) Gobiinae clades (IIB). Our results yield high support for the subfamily Benthophil- M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 inae and its three component tribes, which are the focus of our study, and show less resolution for deeper branches separating other gobioid families. Further research will be necessary to fully identify the arrangement of the Benthophilinae within the higher-order framework of gobioid systematics, which is not our focus. 4.4. Biogeographic patterns Our analysis of divergence times among lineages of Benthophilinae is in general concordance with major geological events in the Ponto-Caspian basin (Fig. 4 and Table 3). The basin has experienced a tumultuous geological history since the mid-Miocene epoch (15 Mya), including multiple large sea-level changes and intermittent connection with the World Ocean, and associated inter-basin connections between the Black and Caspian Sea basins (Mandych, 1995; Reid and Orlova, 2002). These fluctuating water levels and connections caused salinity levels within the basins to range 1–30 ppt over the last 5 My (Reid and Orlova, 2002), resulting in lineage separations on multiple temporal scales. The initial separation of the Black and Caspian Sea basins 5 Mya coincides with the diversification of most Neogobiini + Ponticolini genera (Neogobius, Babka, Mesogobius, Ponticola, and Proterorhinus), as well as diversification within Benthophilini (separation of Benthophilus and Caspiosoma; Fig. 3). Congruently, Cristescu et al. (2003, 2004) identified late Miocene divergences (5.0–7.9 Mya) for benthic amphipods, and Audzijonyte et al. (2008) found a 5 Mya split between Paramysis lineages. These divergences within diverse Ponto-Caspian fauna occurred on a similar time scale as large-scale desiccation events in the Mediterranean Sea basin (Messinian Salinity Crisis 5.9 Mya) and in the eastern Paratethys/early Black Sea basin 5.5 Mya (Hsü and Giovanoli, 1979; Gillet et al., 2007). Desiccation of the Black Sea basin during this period dramatically reduced water levels and increased salinity, enhancing isolation among tributaries within the basin. This led to allopatric separation of taxa residing in these freshwater areas and increased speciation within the more saline basin. In addition to older divergences within Ponto-Caspian taxa, several recent separation events are identified. Onset of the Pleistocene glaciations created additional fluctuations in water levels within the Ponto-Caspian basin (Reid and Orlova, 2002). Several radiation events occurred 1–2 Mya among the Ponticolini, during the midst of these Pleistocene glacial cycles. Notably, in the Ponticola ‘‘platyrostris” species group both Po. cephalargoides and Po. cyrius diverged early 1.82 Mya, and are distributed at opposite ends of the Ponto-Caspian basin today (northwest Black Sea/Azov Sea and the Kura River basin flowing into the Caspian Sea, respectively). Ponticola constructor, Po. platyrostris, and Po. rhodioni then separated 1.3 Mya and are found in the central portion of the Ponto-Caspian basin (marine and freshwater areas of the eastern Black Sea). A similar distribution pattern occurs within the Ponticola ‘‘kessleri” species group; with Po. gorlap occupying marine and freshwater areas of the Caspian Sea basin and Don River, Po. eurycephalus inhabiting marine areas of the northwest Black Sea and Azov Sea, and Po. kessleri primarily found in freshwater drainages of the northwest Black Sea (Dnieper, Dniester and Danube Rivers). The freshwater species of Proterorhinus also occupy an analogous distribution, with Pr. semilunaris occurring in freshwater basins of the northwest Black Sea, Proterorhinus sp. found in the KumoManych Depression (Don/Manych River basin), and Pr. cf semipellucidus inhabiting the upper and lower Volga River basin and delta (Neilson and Stepien, 2009). These three species groups demonstrate a congruent biogeographic pattern: initial isolation and separation of a broadly distributed taxon following closure of an interbasin connection 1.7–2.0 Mya (Apsheron connection through Kumo-Manych Depression; Kaplan, 1995; Reid and Orlova, 2002), succeeded by isolation and further radiation within 95 the Black Sea basin due to glacially-associated fluctuations in water levels and basin shape. In addition, recent water level transgressions and separations within the Pleistocene coincide with lineage divergences of the two subspecies of Neogobius melanostomus (N. m. melanostomus in the Black Sea and N. m. affinis in the Caspian Sea; see Brown and Stepien, 2008; Fig. 3). This pattern of Pleistocene-aged phylogenetic and phylogeographic breaks among Black/Caspian Sea basins is echoed in a variety of taxa ranging from other fishes (Rutilis frisii; Kotlík et al., 2008) to benthic and planktonic aquatic invertebrates (cladocerans—Cristescu et al., 2003, 2004; dreissenid mussels—Stepien et al., 2003, 2002; Gelembiuk et al., 2006; and mysids—Audzijonyte et al., 2006, 2008). Our analysis of divergence times generally is congruent with evolutionary hypotheses proposed for European gobiids, primarily in origins of the ‘‘transverse gobies” (Atlantic-Mediterranean Gobius, Caffrogobius, Nematogobius, Mauligobius, Padogobius, and Zosterisessor) and the ‘‘sand gobies” (Economidichthys, Knipowitschia, and Pomatoschistus; Miller, 2003a). Penzo et al. (1998), using portions of the mt 12S and 16S rRNA genes, estimated a separation time of the transverse and sand gobies of 48 Mya; in the present study using cyt b, we resolved a clade containing Gobius + Zosterisessor (mostly identical to Penzo et al.’s study) separating from a clade containing the sand gobies Knipowitschia and Pomatoschistus 35 Mya (Fig. 3). In addition, McKay and Miller (1997) found a close relationship between the sand gobies and western Pacific gobiids using morphology and isozymes, indicating an earlier separation from the transverse gobies. This association also is seen in our study, with the sand gobies appearing closely related to the western Pacific microdesmids (and ptereleotrids (cyt b; Fig. 3) or to other western Pacific gobiids (COI; Fig. 4). Although Miller (2003a) groups our Ponto-Caspian Benthophilinae as members of the transverse gobiids, it appears that they diverged much earlier from the transverse + sand goby ancestor, 42 Mya. Since the Benthophilinae shares the transverse pattern of cephalic neuromasts with other Atlantic-Mediterranean gobiids, the ‘‘transverse gobies” group is paraphyletic, retaining this ancestral character trait across multiple evolutionary lineages. 5. Conclusion The goby subfamily Benthophilinae represents an understudied yet important component of the Ponto-Caspian fish fauna. We present the most complete phylogenetic and biogeographic study of the group, and clarify outstanding taxonomic issues present for the last 20 years. The Benthophilinae constitutes a unique radiation of gobiid fishes, and is a separate subfamily from the remainder of the Gobiidae. Its evolutionary history has been driven by the dynamic geologic and hydrologic evolution of the Ponto-Caspian basin. 6. Systematic conclusions Benthophilinae Beling and Iljin 1927:309. Type genus Benthophilus Eichwald 1831. Distinguishing features: small to moderate gobiids with infraorbital neuromast organs (comprised of sensory papillae) in 6–7 transverse rows, four before and 2–3 above hyomandibular row b, and lacking row a. Dorsal supraorbital rows o showing separation along dorsal midline. Tubular anterior nostrils, lacking process from the rim. Posterior nostril generally near orbit. Uppermost pectoral fin rays contained within membrane. Swimbladder not present. Moderate to large oligoplasmatic eggs; no pelagic larval stage. Benthophilinae can be separated 96 M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 from the Gobiinae (where it was formerly included) by generally increased number of total (628) and caudal (18–22) vertebrae. Primarily found in the Azov, Black, and Caspian Sea basins and adjacent river drainages; several species introduced into central and northern Europe and the North American Great Lakes. Tribe Benthophilini Beling and Iljin 1927:309. Type genus Benthophilus Eichwald 1831. Benthophilus pinchuki Ragimov 1982. Original name: Benthophilus ctenolepidus pinchuki Ragimov 1982. Benthophilus ragimovi Boldyrev and Bogutskaya 2004. Benthophilus spinosus Kessler 1877. Benthophilus stellatus (Sauvage 1874). Original name: Doliichthys stellatus Sauvage 1874. Synonyms: Benthophilus macrocephalus maeotica Kuznetsov 1888; Benthophilus monstrosus Kuznetsov 1888. Benthophilus svetovidovi Pinchuk and Ragimov 1979. Distinguishing features: small gobiids with infraorbital neuromast organs in 6–7 rows, four before and 2–3 above hyomandibular row b, and lacking row a. Benthophilini can be separated from other members of the Benthophilinae by the combination of complete loss of all head canals, and reduction or complete loss of scales. Genus Caspiosoma Iljin 1927: 129. Type species Gobiosoma caspium Kessler 1877. Included species: Genus Anatirostrum Iljin 1930: 48. Type species Benthophilus profundorum Berg 1927. Included species Tribe Neogobiini new tribe, Neilson and Stepien Type genus Neogobius Iljin 1927. Anatirostrum profundorum (Berg 1927). Original name: Benthophilus profundorum Berg 1927. Genus Benthophiloides Beling and Iljin 1927: 309. Synonym: Asra Iljin 1941: 384. Type species Benthophiloides brauneri Beling and Iljin 1927. Included species: Benthophiloides brauneri Beling and Iljin 1927. Benthophiloides turcomanus (Iljin 1941). Original name: Asra turcomanus Iljin 1941. Genus Benthophilus Eichwald 1831: 77. Synonyms: Bentophilus Eichwald 1838: 102; Hexacanthus Nordmann 1838: 332; Doliichthys Sauvage 1874: 336. Type species Gobius macrocephalus Pallas 1787. Included species: Benthophilus abdurahmanovi Ragimov 1978. Original name: Benthophilus magistri abdurahmanovi Ragimov 1978. Benthophilus baeri Kessler 1877. Benthophilus casachicus Ragimov 1978. Original name: Benthophilus stellatus casachicus Ragimov 1978. Benthophilus ctenolepidus Kessler 1877. Synonym: Benthophilus magistri lencoranicus Ragimov 1982. Benthophilus durrelli Boldyrev and Bogutskaya 2004. Benthophilus granulosus Kessler 1877. Synonym: Benthophilus squamatus Baer in Lukina 1984. Benthophilus grimmi Kessler 1877. Benthophilus kessleri Berg 1927. Original name: Benthophilus grimmi kessleri Berg 1927. Benthophilus leobergius Berg 1949. Original name: Benthophilus stellatus leobergius Berg 1949. Synonym: Benthophilus aculeatus Baer in Lukina 1984. Benthophilus leptocephalus Kessler 1877. Benthophilus leptorhynchus Kessler 1877. Benthophilus macrocephalus (Pallas 1787). Original name: Gobius macrocephalus Pallas 1787. Synonym: Hexacanthus macrocephalus (Nordmann 1838). Benthophilus magistri Iljin 1927. Benthophilus mahmudbejovi Ragimov 1976. Benthophilus nudus Berg 1898. Original name: Benthophilus macrocephalus nudus Berg 1898. Synonym: Benthophilus macrocephalus ponticus Berg 1916. Caspiosoma caspium (Kessler 1877). Original name: Gobiosoma caspium Kessler 1877. Distinguishing features: moderate gobiids with infraorbital neuromast organs in seven rows, four before and three above hyomandibular row b, and lacking row a. Neogobiini can be separated from other members of the Benthophilinae by the following characters: head width about equal to depth; metapterygoid bridge absent; dentary with generally small teeth, largest in the outer row. Genus Neogobius Iljin 1927: 135. Synonyms: Apollonia (subgenus of Gobius) Iljin 1927: 133; Neogobius (subgenus of Gobius) Iljin 1927: 135. Type species: Gobius fluviatilis Pallas 1814. Included species: Neogobius fluviatilis (Pallas 1814). Original name: Gobius fluviatilis Pallas 1814. Synonyms: Gobius sordidus Bennett 1835; Gobius lacteus Nordmann 1840; Gobius stevenii Nordmann 1840; Gobius niger Eichwald 1841 (not of Linnaeus 1758); Gobius fluviatilis nigra Kessler 1859; Gobius fluviatilis pallasi Berg 1916; Gobius caspius Ragimov 1967. Other combination: Apollonia fluviatilis (Stepien and Tumeo 2006). Neogobius melanostomus (Pallas 1814). Original name: Gobius melanostomus Pallas 1814. Synonyms: Gobius cephalarges Pallas 1814; Gobius chilo Pallas 1814; Gobius melanio Pallas 1814; Gobius virescens Pallas 1814; Gobius exanthematosus Pallas 1814; Gobius affinis Eichwald 1831; Gobius sulcatus Eichwald 1831; Gobius lugens Nordmann 1840; Gobius grossholzii Steindachner 1894; Gobius marmoratus Antipa 1909. Other combinations: Gobius melanostomus affinis Navozov 1912; Apollonia melanostomus (Stepien and Tumeo 2006). Neogobius caspius (Eichwald 1831). Original name: Gobius caspius Eichwald 1831. Other combinations: Gobius (Eichwaldia) caspius Smitt 1900; Neogobius (Eichwaldia) caspius (Gaibova 1952). Tribe Ponticolini new tribe, Neilson and Stepien Type genus Ponticola Iljin 1927. Distinguishing features: moderate gobiids with infraorbital neuromast organs in generally seven rows, four before and three above hyomandibular row b, and lacking row a. Ponticolini can be separated from other members of the Benthophilinae by the following characters: metapterygoid bridge present; hyomandibular generally narrow (breadth generally <100% length). M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 Genus Babka Iljin 1927: 132. Synonym: Babka (subgenus of Gobius) Iljin 1927: 132. Type species Gobius gymnotrachelus Kessler 1857. Included species: Babka gymnotrachelus (Kessler 1857). Original name: Gobius gymnotrachelus Kessler 1857. Synonyms: Gobius macropus De Filippi 1863; Gobius burmeisteri Kessler 1877; Gobius macrophthalmus Kessler 1877; Mesogobius gymnotrachelus otschakovinus Zubovitch 1925. Other combinations: Mesogobius gymnotrachelus (Berg 1916); Gobius (Babka) gymnotrachelus Iljin 1927; Gobius (Mesogobius) gymnotrachelus Sözer 1941; Mesogobius gymnotrachelus macrophthalmus (Berg 1949); Gobius (Babka) gymnotrachelus gymnotrachelus Bănărescu 1964; Gobius gymnotrachelus macrophthalmus Ragimov 1967; Neogobius gymnotrachelus (Miller 1973); Neogobius gymnotrachelus gymnotrachelus (Pinchuk 1977); Neogobius gymnotrachelus macrophthalmus (Pinchuk 1977). Genus Mesogobius Bleeker 1874: 317. Synonym: Mesogobius (subgenus of Gobius) Bleeker 1874: 317. Type species Gobius batrachocephalus Pallas 1814. Included species: Mesogobius batrachocephalus (Pallas 1814). Original name: Gobius batrachocephalus Pallas 1814. Synonym: Gobius batrachocephalus borysthenis Pinchuk 1963. Other combinations: Gobius (Mesogobius) batrachocephalus Bleeker 1874; Gobius batrachocephalus batrachocephalus Smitt 1900. Mesogobius nigronotatus (Kessler 1877). Original name: Gobius nigronotatus Kessler 1877. Mesogobius nonultimus (Iljin 1936). Original name: Gobius nonultimus Iljin 1936. Other combination: Mesogobius batrachocephalus nonultimus (Miller 1986). Genus Ponticola Iljin 1927: 134. Synonym: Ponticola (subgenus of Gobius) Iljin 1927: 134. Type species: Gobius ratan Nordmann 1840. Included species: Ponticola bathybius (Kessler 1877). Original name: Gobius bathybius Kessler 1877. Other combinations: Neogobius (Chasar) bathybius (Berg 1949); Neogobius fluviatilis pallasi (Berg 1949); Gobius (Chasar) bathybius Ragimov 1967a; Gobius bathybius Pinchuk 1976; Neogobius bathybius (Pinchuk and Ragimov 1985). Ponticola cephalargoides (Pinchuk 1976). Original name: Neogobius cephalargoides Pinchuk 1976. Synonyms: Gobius syrman Kessler 1859; Gobius constructor Kessler 1874; Gobius cephalarges Chichkoff 1912; Gobius (Ponticola) cephalarges Borcea 1934; Neogobius cephalarges (Georghiev Aleksandrova and Nikolayev 1960); Gobius ratan Pinchuk 1963; Gobius (Ponticola) cephalarges cephalarges Bănărescu 1964; Neogobius ratan (Zambriborshch 1968); Neogobius cephalarges cephalarges (Smirnov 1986). Ponticola constructor (Nordmann 1840). Synonyms: Gobius constructor Nordmann 1840; Gobius platyrostris cyrius Kessler 1879; Gobius platyrostris Berg 1916; Gobius platyrostris cyrius Berg 1923; Gobius cephalarges Iljin (1926) 1927; Gobius (Ponticola) platyrostris cyrius Iljin 1927a; Gobius cephalarges constructor Iljin 1927b; Neogobius cephalarges constructor (Berg 1949); Neogobius platyrostris constructor (Pinchuk 1977). Other combination: Neogobius constructor (Vasil’eva and Vasil’ev 1994). Ponticola cyrius (Kessler 1874). Synonyms: Gobius cyrius Kessler 1874; Gobius weidemanni Kessler 1874; Gobius platyrostris cyrius (Berg 1916); Gobius constructor Berg 1923; Gobius platyrostris 97 Berg 1923; Gobius cephalarges constructor Iljin 1927; Neogobius cephalarges constructor (Berg 1949); Neogobius platyrostris constructor (Pinchuk 1977). Other combination: Neogobius cyrius (Vasil’eva and Vasil’ev 1994). Ponticola eurycephalus (Kessler 1874). Original name: Gobius eurycephalus Kessler 1874. Synonyms: Gobius cephalarges Nordmann 1840; Gobius platyrostris Ul’janin 1871; Gobius constructor Kessler 1874; Gobius (Ponticola) cephalarges Iljin 1927; Neogobius cephalarges (Berg 1949); Gobius cephalarges Pinchuk 1963; Gobius (Ponticola) cephalarges cephalarges Bănărescu 1964; Neogobius platyrostris (Georgiev 1966); Neogobius cephalarges (Bogachik and Remez 1970); Neogobius platyrostris eurycephalus (Pinchuk 1977); Neogobius platyrostris odessicus Pinchuk 1977. Other combinations: Gobius eurycephalus eurycephalus Smitt 1900; Neogobius eurycephalus (Miller 1986). Ponticola gorlap (Iljin in Berg 1949). Original name: Neogobius kessleri gorlap Iljin in Berg 1949. Synonyms: Gobius batrachocephalus Eichwald 1841; Gobius kessleri Kessler 1874; Gobius platyrostris cyrius Derhavin 1926; Gobius cephalarges constructor Derzhavin 1934; Gobius kessleri gorlap Chugunova 1946; Neogobius cephalarges constructor (Berg 1949); Neogobius kessleri Oliva 1960; Neogobius iljini Vasil’eva and Vasil’ev 1996. Other combinations: Neogobius (Ponticola) kessleri gorlap (Gaibova 1952); Gobius gorlap Iljin 1956. Ponticola kessleri (Günther 1861). Original name: Gobius kessleri Günther 1861. Synonyms: Gobius platyrostris Nordmann 1840; Gobius platycephalus Kessler 1857; Gobius cephalarges Steindachner 1870; Gobius batrachocephalus platycephalus Smitt 1900; Gobius trautvetteri Antipa 1909; Gobius (Ponticola) platyrostris Borcea 1934. Other combinations: Gobius (Ponticola) kessleri Iljin 1927; Neogobius kessleri (Berg 1949); Neogobius kessleri kessleri (Pinchuk 1977). Ponticola platyrostris (Pallas 1814). Original name: Gobius platyrostris Pallas 1814. Synonyms: Gobius cephalarges platyrostris Smitt 1900; Gobius cephalarges Smirnov 1959. Other combinations: Gobius (Ponticola) platyrostris Iljin 1927; Neogobius platyrostris (Berg 1949); Neogobius platyrostris platyrostris (Pinchuk 1977). Ponticola ratan (Nordmann 1840). Original name: Gobius ratan Nordmann 1840. Synonyms: Gobius bogdanowi Kessler 1874; Gobius goebelii Kessler 1874; Gobius trautvetteri Kessler 1874. Other combinations: Gobius cephalarges ratan Smitt 1900; Gobius cephalarges bogdanowi Smitt 1900; Gobius cephalarges goebelii Smitt 1900; Gobius rotan Iljin 1927a; Gobius (Ponticola) ratan Iljin 1927b; Neogobius ratan (Berg 1949); Neogobius ratan goebeli (Berg 1949); Neogobius bogdanowi (Berg 1949); Gobius ratan goebeli Iljin 1956; Gobius ratan Pinchuk 1963; Neogobius (Ponticola) ratan ratan (Bănărescu 1964); Neogobius ratan (Zambriborshch 1968); Neogobius ratan ratan (Pinchuk 1976). Ponticola rizensis (Kovačić and Engín 2008). Original name: Neogobius rizensis Kovačić and Engín 2008. Ponticola rhodioni (Vasil’eva and Vasil’ev 1994). Original name: Neogobius rhodioni Vasil’eva and Vasil’ev 1994. Synonyms: Gobius constructor Nordmann 1840; Gobius platyrostris cyrius Kessler 1879; Gobius platyrostris Berg 1923; Gobius platyrostris cyrius Berg 1923; Gobius cephalarges Iljin (1926) 1927; Gobius (Ponticola) platyrostris cyrius Iljin 1927a; Gobius cephalarges constructor Iljin 1927b; Neogobius cephalarges constructor (Berg 1949); Neogobius platyrostris constructor (Pinchuk 1977). Ponticola syrman (Nordmann 1840). Original name: Gobius syrman Nordmann 1840. Synonyms: Gobius trautvetteri Kessler 1859; Gobius eurystomus Kessler 1877; Gobius constructor Borcea 1934. Other combinations: Gobius (Ponticola) syrman Iljin 1927; Neogobius syrman (Berg 1949); Neogobius syrman eurystomus (Berg 1949); Neogobius (Ponticola) syrman eurystomus (Gaibova 1952); Gobius (Ponticola) syrman eurystomus Iljin 98 M.E. Neilson, C.A. Stepien / Molecular Phylogenetics and Evolution 52 (2009) 84–102 1956; Gobius (Ponticola) syrman syrman Bănărescu 1964; Gobius syrman eurystomus Ragimov 1967; Neogobius syrman syrman (Smirnov 1986). Ponticola turani (Kovačić and Engín 2008). Original name: Neogobius turani Kovačić and Engín 2008. Proterorhinus cf semipellucidus (Kessler 1877). Synonyms: Gobius semipellucidus Kessler 1877. Proterorhinus tataricus Freyhof and Naseka 2008. Acknowledgments Genus Proterorhinus Smitt 1900: 544. Synonym: Proterorhinus (subgenus of Gobius) Smitt 1900: 544. Type species Gobius marmoratus Pallas 1814. Included species: Proterorhinus marmoratus (Pallas 1814). Original name: Gobius marmoratus Pallas 1814. Synonyms: Gobius quadricapillus Pallas 1814; Gobius macropterus Nordmann 1840. Proterorhinus nasalis (De Fillipi 1863). Original name: Gobius nasalis De Fillipi 1863. Synonym: Gobius blennioides Kessler 1877. Proterorhinus semilunaris (Heckel 1837). Original name: Gobius semilunaris Heckel 1837. Synonym: Gobius rubromaculatus Kriesch 1873. We thank N. Bogutskaya, V. Boldyrev, L. Corkum, I. Grigorovich, J. Herler, S. Ibrahimov, H. Jenner, J. Kornichuk, V. Kovac, Y. Kvach, A. Naseka, J. Ram, S. Rudnicka, M. Sapoto, P. Simonovic, Y. Slynko, A. Smirnov, and C. Wiesner for specimen collection; V. Boldyrev and E. Vasil’eva for assistance with specimen identification; and D. Murphy for technical advice in the laboratory. This work was funded by a grant from the National Science Foundation (DEB0456972) to C.A.S. We thank J. Banda, J. Brown, A. Haponski, D. Murphy, L. Pierce, R. Lohner, and O. Sepulveda-Villet for valuable comments on the manuscript and N. Bogutskaya, W. Eschmeyer, J. Nelson, and E. Vasil’eva for discussions about the phylogeny. This is publication 2009-005 from the Lake Erie Center. Appendix A Geographic origin, GenBank accession numbers, and specimen ID for outgroup taxa analyzed in the present study. Taxon and author Benthophilus abdurahmanovi Ragimov 1978 B. granulosus Kessler 1877 B. mahmudbejovi Ragimov 1976 B. stellatus Sauvage 1874 Caspiosoma caspium Kessler 1877 Chromogobius zebratus Kolombatović 1981 Gobius bucchichi Steindachner 1870 G. fallax Sarato 1889 G. niger Linnaeus 1758 G. xanthocephalus Heymer and Zander 1992 Pomatoschistus minutus Pallas 1770 Zosterisessor ophiocephalus Pallas 1814 Location Latitude Longitude Specimen GenBank Accession Nos. ID cyt b COI RAG1 S7 Volga River delta, Russia 46.265635 49.093737 AKK5 FJ526777 FJ526832 FJ526886 FJ526960 Volga River delta, Russia Volga River delta, Russia 46.265635 49.093737 AKK7 46.265635 49.093737 AKK10 FJ526778 FJ526779 FJ526833 FJ526834 FJ526887 FJ526888 FJ526961 FJ526962 Dniester River Delta, Bilyayivka, Ukraine Volga River delta, Russia 46.468333 30.216667 ALC6 FJ526780 FJ526835 FJ526889 FJ526963 46.265635 49.093737 AOD3 FJ526781 FJ526836 FJ526890 FJ526964 44.822745 14.337957 AQB2 FJ526783 FJ526838 FJ526892 FJ526966 45.094134 14.436694 AQB6 FJ526784 FJ526839 FJ526893 FJ526967 44.822745 14.337957 AQB8 FJ526785 FJ526840 FJ526894 FJ526968 46.937577 37.258114 AOD8 38.468233 8.986918 AQB10 FJ526782 FJ526795 FJ526837 FJ526841 FJ526891 FJ526895 FJ526965 FJ526969 46.000000 30.500000 AOD1 FJ526776 FJ526831 FJ526885 FJ526959 Adriatic Sea, Isle of Cres, Croatia Adriatic Sea, Isle of Krk, Croatia Adriatic Sea, Isle of Cres, Croatia Yalta Bay, Yalta, Ukraine Atlantic Ocean, Arrabida, Portugal Budaksky Lagoon, Ukraine Black Sea, Odessa, Ukraine 46.470820 30.735090 AHL6 Tyligul Estuary, Ukraine 46.690000 31.486783 AHL4 Kerch Strait, Kerch, 45.358334 36.475834 APT6 Ukraine Appendix B Reference list of taxonomic authorities for all taxa included in the present study. 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