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).
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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-
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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
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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
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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
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FJ526747 FJ526797 FJ526851 FJ526906
EU444670 EU444698 EU444724 FJ526905
FJ526748 FJ526798 FJ526852 FJ526907
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