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New Species of Aphanius (Teleostei, Cyprinodontidae) from Isfahan
Province of Iran and a Reanalysis of Other Iranian Species
TOMAS HRBEK,YAZDAN KEIVANY,AND BRIAN W. COAD
A new killifish species, Aphanius isfahanensis, is described from the Isfahan basin of
Iran. It is distinguished from the other Iranian species of Aphanius by adult color
pattern, molecular character states of mitochondrial DNA sequence data, and in
multivariate morphometric and meristic space. Based on the phylogenetic analysis of
molecular sequence data, the new species is hypothesized to be sister taxontoA.
sophiae plus A. persicus, which also occur in Iran.
THE extant and extinct species of the killifish
genus Aphanius Nardo, 1827 (Cyprinodon-
tiformes) are widely distributed along the late
period Tethys Sea coastlines. The Tethys Sea
closed at the Oligocene/Miocene boundary
(Smith et al., 1995). The distribution includes
coastal areas of the Mediterranean region and
the Gir Peninsula of northwestern India to
northeastern Somalia, including the Red Sea
and the Persian Gulf. Inland distribution is
restricted primarily to the Mediterranean and
the Near East orogenic belt, including Turkey
and Iran. Central Anatolia, Turkey, has been
suggested to be the center of diversity of
Aphanius (Wildekamp et al., 1999). However,
high diversity appears to extend from Turkey to
Iran and is observed in many faunal elements
including cyprinodontoid killifishes (Hrbek et
al., 2002), leuciscine cyprinids (Hrbek et al.,
2004), true salamanders (Weisrock et al., 2001),
and agamid lizards (Macey et al., 1998, 2000).
Distribution and taxonomy of Aphanius in
Anatolia has been subject to several studies
(e.g., Villwock, 1964; Scholl et al., 1978), and
an overview of Aphanius in Turkish waters,
including a discussion on the generic name and
a review of the available literature, is given in
Wildekamp et al. (1999). In contrast to Turkey,
little work has been done on Iranian populations
of Aphanius; however, basic data on reproduc-
tion, alimentation, and habitat of A. vladykovi
exist (Keivany and Soofiani, 2004). Recent
studies (Coad, 1996, 2000) show that, in addition
to the three recognized species of central Iran,
other populations may deserve species recogni-
tion. Specimens from these allopatrically distrib-
uted populations in isolated internal basins
sometimes show color pattern, meristic, or
morphometric differences from other popula-
tions of Aphanius.
The occurrence of marked genetic divergence
among Iranian populations that may or may not
be morphologically distinct resembles the pat-
tern found in the central Anatolian Aphanius
anatoliae species complex (Hrbek et al., 2002).
We therefore suspect that Iran has a much
greater ichthyofaunal diversity than currently
recognized and that this diversity is organized
in accordance with the geological units compris-
ing Iran.
In 2002, we made collections of Aphanius from
the Zagros Mountains of Iran, which harbor
three known species: A. vladykovi,A. sophiae, and
A. persicus. We also collected, from the Zayandeh
River in the vicinity of Isfahan, Iran (Fig. 1),
a suspected new species of Aphanius that differs
in pigmentation pattern from all other Iranian
species. We obtained morphometric, meristic,
molecular, and distributional data, ultimately
concluding that the Zayandeh River basin con-
tains a species previously unknown to science.
Fig. 1. Map of collections of Iranian Aphanius
made for this study. Base map was created at Online
Map Creations http://www.aquarius.geomar.de/
omc/.
Copeia, 2006(2), pp. 244–255
#2006 by the American Society of Ichthyologists and Herpetologists
MATERIALS AND METHODS
Measurements and counts follow Coad (1988,
1996). A set of 20 standardized measurements
(Table 1) and eight counts (Table 2) was
collected for all specimens. Measurements, in-
cluding sub-units of head, are presented as
percentages of standard length (SL) in Table 2.
Cross-bar count (Table 2) was taken by counting
the number of dark bars (males only) on the
mid-lateral series of scales on the left side. To
avoid potentially confounding effects of allome-
try (Atchley et al., 1976), the raw morphometric
data were log-transformed and then transformed
into residuals using the least-squares regression
method (Sokal and Rohlf, 1994). Meristic data
were analyzed untransformed. The morphomet-
ric residuals and meristic characters were ana-
lyzed using General Linear Model (GLM) as
implemented in SYSTAT 10.2.05 (Systat Soft-
ware, Inc.), where species and sex and their
interaction effect were the independent vari-
ables. Multivariate data were further summarized
and visualized using the principal component
analysis (PCA) implemented in SYSTAT 10.2.05
(Systat Software, Inc.). The PCA of morphomet-
ric data were performed for each sex separately,
but sexes were analyzed jointly in meristic PCA.
Institutional abbreviations follow Leviton et al.
(1985) with the addition of CMNFi, Canadian
Museum of Nature, Ottawa, Canada.
Molecular analyses.—We obtained mitochondrial
DNA sequence data from 18 individuals repre-
senting four species. Aphanius asquamatus,A.
fasciatus, and the central Anatolian Aphanius
anatoliae species complex were used as the
outgroup data set. Choice of outgroup taxa was
based on previous molecular studies of Aphanius
(Hrbek et al., 2002; Hrbek and Meyer, 2003).
DNA sequences included genes encoding a por-
tion of 12S ribosomal RNA, complete sequences
of NADH1 and NADH2, transfer RNAs coding
for valine, leucine, isoleucine, glutamine, methi-
onine, tryptophan, alanine, asparagine, cysteine,
and tyrosine, and the light-strand replication
origin. Amplification and sequencing primers
and sequencing strategy are described by Hrbek
and Meyer (2003).
In all instances, the two individuals sequenced
per population were identical or nearly identical;
therefore, we randomly selected one of the two
individuals for all subsequent analyses. Maximum
parsimony and maximum likelihood phylogenet-
ic relationships were estimated using the pro-
gram PAUP* v4.10b (D. L. Swofford, PAUP*:
phylogenetic analysis using parsimony [*and
other methods], Sinauer, Sunderland, MA,
2002). Bayesian likelihood phylogenetic relation-
ships were estimated in the program MrBayes
3.0b4 (Ronquist and Huelsenbeck, 2003). Maxi-
mum likelihood estimates were done under the
GTR model of evolution (Rodriguez et al., 1990)
with rates for variable sites assumed to follow the
gamma distribution and with a portion of sites
assumed to be invariable; this model of molecu-
lar evolution was indicated by the program
MODELTEST (Posada and Crandall, 1998) as
best fitting the data. Sequence data for new
Iranian populations have been deposited in
GenBank under numbers AY593481–AY593498.
Previously published sequence information is
from Hrbek and Meyer (2003). Aligned se-
quences are available from the first author.
Aphanius isfahanensis, new species
Kapourdandan-e Isfahan (Farsi)
Figure 2A, B
Holotype.—CMNFi2004-0001, male, 25.0 mm SL,
Iran, Isfahan Province, Zayandeh Rud (Zayandeh
River) at Varzaneh Bridge, 32u259320N,
52u399140E, 1 July 2002, Y. Keivany and S.
Asadollah.
Paratypes.—CMNFi2004-0002, 18 males, 20.8–
30.9 mm SL, 18 females, 22.0–38.4 mm SL, of
49 total (13 not used in meristic and morpho-
metric analyses), same locality as holotype;
AMNH 233639, 1 male, 25.2 mm SL, 1 female,
21.6 mm SL, same locality as holotype; MRAC
2004-05-P-01-02, 1 male, 25.2 mm SL, 1 female,
21.6 mm SL, same locality as holotype; GenBank
accession numbers AY593488, AY593489,
AY593497, and AY593498.
Diagnosis.—Aphanius isfahanensis is clearly distin-
guished at the genetic level from all other species
of Aphanius. It has 82 molecular apomorphies–19
transversions, two transversions/transitions (de-
pending on comparison), and 61 transitions–that
show fixed character state differences to homol-
ogous characters analyzed in A. sophiae,A.
persicus, and A. vladykovi from Iran (Table 3).
Thirty-seven of these character states are also
apomorphies when compared to A. anatoliae,A.
danfordii,A. villwocki,A. asquamatus,andA.
fasciatus from Turkey. Alignment with apomor-
phies highlighted in yellow is available at
www.cyprinodontiformes.org.
Males of A. isfahanensis (Fig. 2a) can be
distinguished from those of all other Iranian
species by having distinct black edge on the
dorsal, anal, and pelvic fins. The dorsal fin is
HRBEK ET AL.—NEW APHANIUS FROM IRAN 245
TABLE 1. MORPHOMETRIC CHARACTERS OF THE HOLOTYPE AND ADDITIONAL SPECIES OF Aphanius OF THE EASTERN CLADE (HRBEK AND MEYER, 2003) FROM IRAN.n5number of
specimens; each cell contains mean 6standard deviation and range (minimum2maximum).
m
5male;
f
5female;
s
5significant pair-wise difference between A. isfahanensis and
A. sophiae;
p
5significant pair-wise difference between A. isfahanensis and A. persicus;
v
5significant pair-wise difference between A. isfahanensis and A. vladykovi; cut-off P50.0025.
Character Holotype =A. isfahanensis =
(n518) A. isfahanensis R
(n519) A. sophiae =
(n540) A. sophiae R
(n534) A. persicus =
(n5177) A. persicus R
(n5228) A. vladykovi =
(n536) A. vladykovi R
(n516)
Standard length 25 25.8 63.29 29.2 65.36 26.7 64.49 29.2 64.84 22.8 63.88 26.0 65.79 27.0 64.53 29.1 65.13
(20.8–30.9) (22.0–38.4) (18.1–39.3) (20.0–42.0) (15.9–33.1) (15.7–45.9) (21.6–36.6) (23.5–40.2)
Body depth 31.60 32.05 61.91 29.98 62.81 31.25 62.36 29.71 62.37 32.06 62.06 30.88 61.75 33.18 61.70 31.94 61.18
(28.82–36.49) (25.41–35.94) (25.97–36.66) (25.95–36.48) (27.32–37.92) (26.18–37.00) (29.30–36.16) (30.12–33.83)
Head
depth
ms,mv,fp
27.20 27.03 61.40 25.04 61.06 24.70 61.27 23.84 61.63 26.54 61.52 25.91 61.44 25.70 61.04 25.18 60.94
(23.97–30.07) (23.43–27.37) (22.55–27.44) (21.20–28.34) (23.39–30.64) (22.34–30.25) (23.48–28.62) (23.94–27.11)
Head
length
ms,fp,fv
30.40 31.20 61.19 29.24 60.81 29.08 61.00 28.16 61.47 30.90 61.24 30.70 61.49 31.13 60.94 30.48 61.16
(29.45–34.31) (27.94–31.08) (27.74–31.28) (25.26–31.00) (27.43–34.34) (27.05–34.56) (29.00–32.88) (28.96–33.08)
Head width
ms
20.40 20.90 61.03 21.14 60.60 18.87 60.88 19.07 61.30 20.37 61.07 20.77 61.47 20.36 60.73 20.84 60.85
(19.42–22.55) (20.06–22.26) (17.12–21.20) (15.74–21.50) (17.94–24.26) (17.87–25.81) (18.83–21.88) (19.69–23.13)
Snout
length
ms,mv
8.00 8.59 60.61 8.12 60.66 7.47 60.43 7.44 60.63 8.28 60.81 8.34 60.94 8.12 60.55 7.95 60.72
(7.92–10.46) (7.19–9.60) (6.63–8.40) (6.44–9.15) (6.36–10.44) (6.12–12.04) (7.33–9.32) (7.23–9.95)
Mouth
width
ms,mv,fs
10.40 10.81 60.87 10.59 60.78 9.32 60.71 9.07 60.92 11.05 61.07 11.27 61.47 9.86 60.68 9.89 60.82
(9.59–12.68) (8.92–11.72) (8.36–11.45) (7.48–11.49) (8.44–14.87) (8.33–17.65) (8.54–11.56) (8.75–11.44)
Orbit
diameter
ms,fp,fv
9.20 9.89 60.64 8.68 60.58 9.18 60.56 8.82 60.48 10.05 60.57 9.40 60.59 9.91 60.62 9.52 60.58
(8.74–11.21) (7.69–9.70) (8.33–10.13) (7.87–9.77) (8.55–11.60) (7.91–11.88) (8.67–11.34) (8.46–10.42)
Interorbital
width
mp,fp
11.60 12.20 60.68 11.90 60.87 11.81 60.57 11.20 60.75 12.71 60.70 12.49 60.83 12.40 60.73 12.18 60.77
(11.11–13.70) (10.16–13.54) (11.00–13.22) (9.80–13.03) (10.78–15.15) (10.19–15.16) (10.99–13.74) (10.94–13.62)
Postorbital
length
mp,mv,fp,fv
13.60 13.46 60.57 13.65 60.71 13.31 60.76 13.08 60.91 13.75 61.00 14.32 61.24 14.81 60.88 14.79 61.18
(11.87–14.53) (12.50–15.41) (11.68–14.66) (11.84–14.92) (11.54–16.36) (11.64–18.01) (13.33–16.47) (12.92–17.91)
Predorsal
length
mp,mv,fp,fv
59.60 58.35 61.71 61.38 62.27 59.07 61.81 60.10 61.52 60.96 61.39 62.14 61.63 64.99 61.47 65.68 61.51
(54.71–60.75) (57.14–64.60) (55.74–62.82) (57.74–63.19) (56.71–64.60) (57.38–66.43) (62.17–67.83) (62.81–69.15)
Dorsal length
mv
27.60 28.03 61.70 22.53 60.99 29.15 62.63 23.22 61.78 28.75 62.46 23.32 61.48 25.34 61.58 21.57 61.08
(23.61–31.05) (20.62–24.59) (23.20–33.92) (20.75–27.06) (22.16–34.41) (19.62–26.54) (21.90–29.90) (20.00–23.40)
Prepelvic
length
fs,fp
53.60 54.15 63.06 57.10 62.65 52.56 61.91 53.37 62.52 54.03 61.84 55.00 62.05 54.50 61.38 54.97 61.35
(50.00–59.87) (51.36–62.16) (49.03–57.14) (48.93–59.52) (48.40–59.51) (49.31–63.99) (51.84–57.34) (52.77–58.46)
Preanal length
fs
64.40 65.72 62.33 69.85 61.99 65.90 61.41 67.29 61.87 66.68 61.83 68.25 62.02 66.26 64.05 68.47 60.99
(62.04–69.93) (65.77–73.02) (63.18–68.55) (64.56–70.57) (61.75–72.54) (63.59–78.78) (43.33–69.23) (66.38–70.00)
Anal length
mv
22.40 22.65 61.26 19.16 61.11 23.07 61.60 19.59 61.30 22.51 61.61 23.32 61.48 21.21 61.08 19.38 61.05
(20.49–24.84) (16.80–20.83) (19.35–26.67) (16.82–22.44) (18.05–26.98) (19.62–26.54) (18.18–24.44) (17.89–22.13)
Caudal peduncle
length
mp,mv
25.20 25.33 60.91 23.59 61.46 24.74 61.32 24.27 60.93 23.68 61.02 23.08 61.22 23.23 61.03 22.65 60.63
(23.65–27.54) (20.36–25.77) (22.34–27.48) (22.41–25.87) (20.65–26.41) (18.37–26.70) (21.54–25.48) (21.57–24.21)
246 COPEIA, 2006, NO. 2
Character Holotype =A. isfahanensis =
(n518) A. isfahanensis R
(n519) A. sophiae =
(n540) A. sophiae R
(n534) A. persicus =
(n5177) A. persicus R
(n5228) A. vladykovi =
(n536) A. vladykovi R
(n516)
Caudal peduncle
depth
17.20 17.75 60.94 15.30 60.85 17.39 61.29 15.93 60.85 16.97 61.23 15.43 61.00 17.31 60.82 15.78 60.48
(16.10–19.59) (13.84–16.73) (15.08–19.92) (14.21–17.62) (13.36–20.08) (13.11–18.44) (15.51–19.21) (15.09–16.92)
Pectoral fin
length
mp,fp
17.20 17.23 61.21 15.79 61.12 18.23 61.19 16.87 61.41 19.26 61.39 17.23 61.37 17.80 60.85 16.23 61.13
(14.84–18.96) (13.06–17.84) (16.02–21.59) (14.64–20.13) (15.49–23.48) (13.58–21.76) (15.84–19.91) (13.11–18.16)
Pelvic fin
length
ms,mp,fs,fp
6.40 6.89 60.82 6.80 60.61 9.78 61.05 8.82 60.87 7.88 61.13 7.59 60.94 7.48 60.77 7.21 61.14
(5.71–8.45) (5.65–8.09) (6.63–12.00) (7.33–10.56) (2.35–11.93) (4.63–10.45) (5.63–9.04) (5.42–9.51)
Pectoral–pelvic fin
distance
ms,mp,mv,fs,fp,fv
25.20 24.73 61.86 28.18 62.39 22.80 61.47 25.25 62.96 22.63 61.49 24.29 62.02 23.33 61.30 25.10 61.22
(21.49–28.62) (23.64–31.20) (18.29–25.19) (20.17–34.18) (18.77–28.57) (19.43–31.22) (20.44–26.01) (22.55–27.22)
Pelvic–anal fin
distance
ms,mp
10.80 11.26 61.33 13.33 60.79 13.35 61.25 13.80 61.55 12.26 61.14 12.94 61.18 12.44 61.05 13.95 61.01
(8.17–14.02) (12.31–14.77) (10.50–15.97) (9.48–17.17) (9.27–16.92) (10.27–16.48) (10.17–15.11) (12.45–15.94)
TABLE 1. CONTINUED.
TABLE 2. MERISTIC CHARACTERS OF THE HOLOTYPE AND ADDITIONAL SPECIES OF Aphanius OF THE EASTERN CLADE (HRBEK AND MEYER, 2003) FROM IRAN.n5number of specimens;
each cell contains mean 6standard deviation and range (minimum–maximum).
s
5significant pair-wise difference between A. isfahanensis and A. sophiae;
p
5significant pair-
wise difference between A. isfahanensis and A. persicus;
v
5significant pair-wise difference between A. isfahanensis and A. vladykovi; cut-off P50.0063.
Character Holotype =A. isfahanensis =
(n518) A. isfahanensis R
(n519) A. sophiae =
(n540) A. sophiae R
(n534) A. persicus =
(n5177) A. persicus R
(n5228) A. vladykovi =
(n536) A. vladykovi R
(n516)
Gill rakers
s,p,v
13 12.2 60.62 12.3 60.89 11.4 60.70 11.3 60.72 11.2 60.73 11.2 60.86 10.9 60.52 11.0 60.37
(11–13) (10–13) (10–13) (10–12) (9–14) (9–14) (10–12) (10–12)
Lateral-line scales
s,v
28 26.8 61.10 27.4 60.77 28.3 61.26 28.6 61.07 26.9 61.01 27.1 61.06 39.4 62.35 39.0 61.86
(25–28) (26–29) (27–31) (25–31) (24–29) (24–30) (36–47) (36–43)
Precaudal vertebrae
v
11 11.4 60.61 12.0 60.58 12.0 60.66 12.0 60.58 — — 12.0 60.69 12.2 60.40
(10–12) (11–13) (11–13) (11–13) (10–13) (12–13)
Caudal vertebrae 17 16.2 60.71 15.9 60.88 16.3 60.63 16.3 60.63 — — 16.5 60.56 16.3 60.45
(15–17) (15–18) (15–17) (15–17) (15–17) (16–17)
Pectoral-fin rays
s
16 15.4 60.62 15.1 60.91 16.0 60.82 16.1 61.04 15.0 60.79 15.0 60.85 15.7 60.78 15.4 60.81
(14–16) (13–16) (14–18) (15–19) (13–17) (11–18) (14–17) (14–17)
Pelvic-fin rays
s,v
6 5.3 60.58 5.5 60.51 5.8 60.41 5.6 60.49 5.4 60.59 5.5 60.54 5.9 60.52 5.9 60.50
(4–6) (5–6) (5–6) (5–6) (3–6) (4–6) (5–8) (5–7)
Dorsal-fin rays
s
13 12.2 60.71 12.1 60.88 13.1 60.85 13.0 60.76 11.9 60.86 12.0 60.77 12.5 60.61 12.4 60.81
(11–14) (11–14) (12–15) (11–15) (8–14) (10–14) (11–14) (11–13)
Anal-fin rays
s,p,v
12 11.2 60.51 11.3 60.67 11.8 60.79 11.8 60.64 10.9 60.71 10.9 60.69 12.1 60.72 11.9 60.77
(10–12) (10–13) (10–13) (11–13) (9–13) (9–13) (11–13) (11–13)
Male flank bars
s,v
11 10.2 60.99 — 14.4 62.52 — — — 11.5 61.48 —
(8–12) (10–21) (9–14)
HRBEK ET AL.—NEW APHANIUS FROM IRAN 247
covered with a high density of black blotches.
However, this color pattern is also observed in
males of Aphanius anatoliae from Turkey.
Females of A. isfahanensis (Fig. 2b) can be
distinguished from females of A. sophiae and A.
vladykovi by having flank-bars rather than spots. It
can also be distinguished from A. persicus by less
well-defined bars terminating at a mid-flank
stripe and a relatively light gray stripe at the
caudal-fin base rather than a black spot or blotch.
Flank-bars are also characteristic of females of
Aphanius dispar and Aphanius fasciatus, however.
Aphanius isfahanensis can be distinguished
unambiguously only from A. vladykovi by a lower
lateral-line scale count. It cannot be distin-
guished unambiguously by any morphometric
or meristic character from A. sophiae or A.
persicus.
Description.—Morphometric and meristic charac-
ters are summarized in Tables 1 and 2 and
Figure 3. Snout rounded, 26%head length.
Lower jaw directed upward at 30uangle, poste-
rior tip of maxilla halfway between snout and
edge of orbit. Head depth and head length is on
average relatively larger, but head width relatively
smaller than in other Iranian species. Eye 30%of
head length, and in males the eye relatively
smaller than in other Iranian species. Body depth
approximately 32%body length. Head profile
straight and dorsal profile rounded. Anal fin with
superior border curved, pectoral fin rounded
and inserted at below midline of body, ventral
fins short and inserted just anterior to anal fin,
caudal fin rounded. Total vertebrae 28. Males
bulky and high-bodied, reaching 31 mm SL.
Greatest body depth just anterior of pelvic fins.
Females larger than males, reaching over 38 mm
SL. Body and fin morphology identical to that of
males, but females have relatively short pelvic fins
and relatively long belly relative to other Iranian
species. Anus and oviduct separated.
Pigmentation.—Counts of male flank bars are
given in Table 2; there are significantly fewer
flank bars than in A. sophiae. The bars are broad
with interspaces about equal or slightly narrower
(Fig. 2a). The bars extend from behind the head
to the tail. Anterior bars fade on the belly,
whereas, posteriorly on the caudal peduncle,
they encircle the body. Dorsally, the head is dark
gray and the body is lighter but still heavily
pigmented with melanophores; the belly lacks
pigmentation. The sides of the head are densely
speckled with melanophores, more thinly on the
ventral side; in most specimens the chin is darker
than the rest of the ventral head surface. The eye
is bounded ventrally and postero-ventrally by
a thin line of black pigment. The defining male
coloration is the black margins of the pelvic,
anal, and dorsal fins. The dorsal and anal fins
may present a halo effect, the margins being so
dark in relation to the rest of these fins. The tips
and outer margin of the pelvic fin are blackish.
The anal fin has a broad, blackish margin with
the rest of the fin light cream-colored. The dorsal
fin has the blackest margin. The rest of the dorsal
fin is variably blotched, the blotches being much
lighter than the fin margin. Most specimens have
a contrasting pigmentless area just below the fin
margin. The pectoral fin has sparse pigmentation
along rays and ventrally on the interradial
membranes but lacks the concentrated black
pigmentation seen on the pelvic fins. The caudal-
fin rays and membranes are sparsely pigmented,
and the whole margin may be blackish but in
most fish pigment is restricted to the upper and
lower margins, the lower margin only, or is
absent.
Large females have a grayish dorsal surface on
the head, a lighter back and upper flank covered
with scattered melanophores, and a mid-flank
Fig. 2. (A) Aphanius isfahanensis, male holotype, 25.0 mm SL. (B) Aphanius isfahanensis, female paratype,
38.3 mm SL from the type locality.
R
Fig. 3. Principal component analysis of 20
standardized morphometric measurements in males
(A) and females (B) and of six meristic counts
analyzed jointly for both sexes (C). Open circles
(#) represent specimens of A. isfahanensis, vertical
crosses (+) represent A. sophiae, diagonal crosses
(3) represent A. persicus, and triangles (m) repre-
sent A. vladykovi. Ellipses denote the 68.27%density
limits of each sample.
248 COPEIA, 2006, NO. 2
TABLE 3. SUMMARY OF 82 MOLECULAR CHARACTERS WITH FIXED APOMORPHIC STATES IN A. isfahanensis. Of the 82 molecular apomorphies, 19 are transversion, two are transversions/
transitions (depending on comparison), and 61 are transitions. Thirty-seven of these character states remain apomorphic when comparison is expanded to include A. anatoliae,A.
danfordii,A. villwocki,A. asquamatus, and A. fasciatus from Turkey. No variation in character state exists within any of these species. Numbers above characters indicate the char-
acter’s position in the complete 3303 molecular character matrix. See GenBank (www.ncbi.nlm.gov) or www.cyprinodontiformes.org for complete data matrix.
111111111111111111111112222222222222222222222222222222222222333
1244777777888899999000111222233334444567990000011111111222223445555556666788899011
8028015699147911677356667137723891358449171555722367888124555280046792479123855219
1801845009411639539643351170643791247084587015425110258840147823940033452013457681
A. isfahanensis AGGATCTAGAGTAATCTTTCCACTAATTAGCGTGTACCATCCCTTTTAGGTGAACGTTTTAAGCGGTACGATAGCGGTAGCA
A. persicus GAAGCACCAGACTCCTCCATTGTGTGCCCATAGACTTAGCGTTCCCCGTAAATGTAGCCCGGATACCGAACCCATAACGAAG
A. sophiae GAAGCACCAGACTCCTCCATTGTATGCCCATAGACTTAGCATTCCCCGTAAATGTAACCCGGATACCGAACCCATAACGAAG
A. vladykovi GAAGCACCAGACTCCTCCATTGTACGCCCATACACTTAGCATTCCCCGTAAATGTAGCCCGGATAACGAATCCATAACGAAG
HRBEK ET AL.—NEW APHANIUS FROM IRAN 249
stripe terminating on the caudal peduncle in
a blackish, short stretch covering up to three
scales (Fig. 2b). This short stretch of pigment is
present in all females, faint in some, rarely
forming a blotch and in some small fish tapering
anteriorly. The flank stripe may be broken into
a series of blotches in some smaller females, or it
may be continuous as in large fish. Starting
anterior to the belly there is a ventrolateral series
of thin bars (up to ten) separated by cream-
colored interspaces 1–3 bars wide. These are
absent in some smaller females, which may only
have blotches at various levels presenting this
region. Even some large females have faint flank
pigmentation so that bars and the stripe are
weakly expressed. At about the origin of the anal
fin, the flank bars may continue onto the caudal
peduncle in regular form or become irregular,
breaking up into blotches. Anteriorly the bars
terminate ventrally at about the level of the lower
edge of the pectoral fin. The belly and lower
head have sparse pigment although the chin and
sides of head are speckled with melanophores.
The eye is bounded ventrally and postero-
ventrally by a thin line of black pigment. Fins
lack any distinctive pigment pattern. Fin rays are
outlined with melanophores, and interradial
membranes of the caudal and anal fins have
melanophores at varying degrees of density. The
dorsal fin has the most interradial pigmentation,
particularly near, but clear of the fin base.
Distribution and habitat.—The type locality (Fig. 1)
is near the town of Varzaneh on the lower
reaches of the Zayanadeh River, about 30 km
upriver from the terminal sump, the Gav Khuni
marsh. Water temperature was 27 C, pH 6.7,
water was brackish, conductivity 10.9 mS, dis-
solved solids 5450 ppm, dissolved oxygen
12.3 mg/L, river width was 50 m, and capture
depth 0.5 m. Current was slow, and there was no
cover. Gambusia holbrooki was captured at the
same locality. Aphanius isfahanensis appears to be
restricted to the internal Zayanadeh River basin.
Phylogenetic relationships.—The genus Aphanius is
divided into two major clades, the western and
the eastern clade (Hrbek and Meyer, 2003). The
western clade contains the main radiation found
in the orogenic belt from central Anatolia,
Turkey to southern Iran. This radiation also
contains the Mediterranean brackish-water spe-
cies A. fasciatus. The Iranian group is mono-
phyletic and forms the sister group to A. fasciatus
+A. asquamatus +A. anatoliae species complex of
central Anatolia. A total of 3303 characters were
included in the analyses; 1012 were variable and
697 were parsimony informative; data showed no
visible effect of saturation. The parsimony
phylogeny has a CI of 0.650 and RI of 0.747.
The maximum-likelihood phylogeny (Fig. 4) is
Fig. 4. Maximum likelihood estimate of phylo-
genetic relationships of Aphanius isfahanensis to
other species of Aphanius of the western clade of
species of Aphanius (Hrbek and Meyer, 2003). -ln L
514164.01742, a50.950747, pinvar 50.538377.
Numbers above nodes represent Bayesian likeli-
hood values followed by maximum likelihood and
maximum parsimony bootstrap values. Species and
geographic localities correspond to those listed in
Material Examined and those from Hrbek and
Meyer (2003). Localities of Aphanius sophiae are: (1)
Dolarabad, Iran; (2) Malaskuh, Iran; (3) Abdol-
mahdi, Iran; and (4) Kor River basin near Shiraz,
Iran. Aphanius persicus:NasrabadSpringnear
Imamzadeh Ibrahim, Iran. Aphanius vladykovi: (1)
Boldaji, Iran; (2) Nadar-Bokhtri, Iran; (3) Ebrahi-
mabad, Iran; and (4) Taqanak, Iran. Aphanius
anatoliae anatoliae : (1) Lake Eg˘irdir at Karaot,
Turkey; (2) Lake Isıklı near Beydilli, Turkey; and
(3) Thaliye Canal by Sulamasi, Turkey. Aphanius
anatoliae splendens: Lake Salda, Turkey. Aphanius
danfordii: Soysalii springs in Sultan Swamps, Turkey.
Aphanius villwocki: Pinarbas¸i in upper Sakarya River,
Turkey. Aphanius fasciatus: Lake Tuzla near Tarsus,
Turkey. Aphanius asquamatus: Lake Hazer, Turkey.
Both individuals of Aphanius isfahanensis are from
Zayandeh River near Varzaneh, Iran.
250 COPEIA, 2006, NO. 2
identical to the maximum parsimony phylogeny;
parameters are -ln L 514201.78889, a5
0.800049, invar 50.504877. Phylogenetic analy-
ses support the hypothesis that A. isfahanensis is
sister to A. sophiae from the Kor basin +A. persicus
from the Maharlu basin (Fig. 4); sister to these
three taxa is A. vladykovi from the highland
(.2000 m) plains of the Kukalar mountains in
the upper reaches of the Karun River drainage
system (Fig. 1). All species are monophyletic and
clearly divergent. Hrbek and Meyer (2003)
estimated that A. sophiae and A. vladykovi di-
verged from each other approximately five
million years ago. These estimates were based
on several geological (Bender, 1968; Krijgsman
et al., 1999) and fossil (Sauvage, 1874; Gaudant,
1978; Reichenbacher and Weidmann, 1992)
calibration points. Using these same calibrations
points, A. isfahanensis is estimated to have di-
verged approximately 4.8 million years ago from
a lineage leading to A. sophiae and A. persicus.
Etymology.—Named after the province and its
capital city, Isfahan, central Iran.
DISCUSSION
The geological history of Iran is similar to
Anatolia in that it has been shaped by geological
events associated with the closing of the Tethys
Sea. Detailed sampling and analysis of central
Anatolian populations of Aphanius revealed at
least seven deeply divergent (Hrbek et al., 2002),
and in cases where investigated, reproductively
isolated (Villwock, 1964) lineages, but only three
scientifically recognized species (Wildekamp et
al., 1999; Hrbek and Wildekamp, 2003). All
lineages investigated are allopatrically distributed
in separate hydrological basins.
Similar to Anatolia, rapid isolation of multiple
areas in Iran was caused by northward movement
of the Arabian plate into the Iranian plate
starting ten MYA (Dercourt et al., 1986). The
initial phase caused the uplift of the Zagros
Mountains at the southern edge of the Iranian
plate. Continued north-eastern movement of the
Arabian plate and a northerly movement of India
resulted in additional mountain building by five
MYA (Dercourt et al., 1986) along the northern
edge of the Iranian plateau as well as along the
sutures of the Iranian, Lut, Helmand, and Farah
plates which comprise present-day Iran. If geo-
logical events had a similar effect on the fauna of
Iran as did geological events on Anatolian taxa
(Weisrock et al., 2001; Hrbek et al., 2002, 2004),
we would expect the various populations of
Aphanius occurring in isolated basins of the
Zagros, Little Caucasus, and the Elburz moun-
tains and intervening Iranian plateau to form
monophyletic groups. Due to the rapid and
nearly simultaneous isolation of these areas from
one another, we also would expect relationships
among these populations to approximate a hard
polytomy.
Until this study, relatively little attention has
been paid to the role of geological events in
generating the biological diversity of Iran. To our
knowledge, only in the case of agamid lizards
(Macey et al., 1998, 2000; Rastegar-Pouyani and
Nilson, 2002) have geological events been
implicated and tested as the causal mechanism
in faunal diversification. However, geological
events associated with the accretion of present-
day Iran also seem to play a role in generating
biodiversity of Aphanius. Based on estimates of
five MYA divergence of A. sophiae and A. vladykovi
(Hrbek and Meyer, 2003) derived from several
independent geological (Bender, 1968; Krijgs-
man et al., 1999) and fossil (Sauvage, 1874;
Gaudant, 1978; Reichenbacher and Weidmann,
1992) calibration points, we estimate a 4.8 MYA
divergence of A. isfahanensis and A. sophiae +A.
persicus. This divergence time is in accord with
our hypothesis of a near-simultaneous diversifi-
cation of ,5 MYA of organisms occupying
different geological units of central Iran.
If this pattern is corroborated, and if it reflects
the pattern observed in Anatolia, then Iran
probably harbors numerous other species in
addition to those currently recognized. Four
species, including Aphanius isfahanensis,are
presently recognized from central Iran, but
Aphanius lives in at least two additional geo-
logically and hydrologically isolated internal
basins (Coad, 2000). Additional studies probably
will result in recognition of greater Iranian fish
diversity than currently appreciated, as well as in
the biodiversity of other organismal groups.
A noticeable feature of the ichythyofauna of
the Near East is the relative high degree of
conservatism of external morphology in spite of
substantial genetic, and when studied, reproduc-
tive (Villwock, 1964) divergence. In our analysis
of the Iranian members of the eastern clade of
Aphanius there are no fixed external morpholog-
ical characters that would distinguish A. isfaha-
nensis from its closest relatives A. sophiae and A.
persicus.Aphanius isfahanensis can be distin-
guished by non-overlapping lateral-line scale
number from A. vladykovi, the fourth Iranian
member of this clade. Aphanius isfahanensis,A.
sophiae, and A. persicus are not distinguishable
qualitatively and difficult to distinguish even
quantitatively by multivariate analyses of mor-
phological data (Table 4, 5; Fig. 3a, b, c) but are
clearly distinguishable by molecular data
HRBEK ET AL.—NEW APHANIUS FROM IRAN 251
(Table 3; Fig. 4). Species of Aphanius also often
differ in male color pattern, differences poten-
tially attributable to sexual selection, which is
major evolutionary force in killifishes (e.g.,
Fuller, 2002). However, these color patterns
often appear to be convergent among species
of Aphanius, a phenomenon clearly observed in
other taxa (West-Eberhard, 2003; Coyne, 2004).
Although taxonomically not satisfactory, knowl-
edge of collecting locality or drainage basin is the
most practical information for rapid species
identification, followed by color pattern, if
approximate collection data are known. Howev-
er, only molecular analyses can unambiguously
distinguish among different species of Aphanius.
This and our previous studies (Hrbek et al.,
2002, 2004) suggest that, in active orogenic areas
with opportunities for allopatric speciation,
much of the extant biodiversity remains and will
remain unrecognized under many species con-
cepts. Allopatrically distributed species are often
very similar in external morphological and
meristic characters and, thus, difficult to distin-
guish, as should be evident from this study. This
TABLE 4. SUMMARY OF MORPHOMETRIC CHARACTER LOADINGS FOR PCA SEPARATED BY SEX OF A. isfahanensis,A. sophiae,
A. persicus,AND A. vladykovi AND PERCENT VARIANCE EXPLAINED BY EACH EIGENVECTOR. First three eigenvectors explain
over 50%of total variance. Effects of size were removed by converting log-transformed morphometric data into
residuals using the least-squares regression method. Heavy loadings (.75%) are indicated in bold.
Character
Eigenvector
Female Male
1 23123
Body depth 0.713 0.038 20.112 0.558 20.207 0.008
Head depth 0.843 0.060 0.077 0.872 20.097 0.011
Head length 0.847 20.101 20.077 0.899 0.035 20.120
Head width 0.739 20.300 0.137 0.794 20.218 20.053
Snout length 0.704 0.040 0.160 0.732 0.050 20.229
Mouth width 0.587 20.033 20.070 0.559 0.157 0.025
Orbit diameter 0.693 0.071 20.062 0.814 0.168 20.165
Interorbital width 0.683 20.188 20.366 0.838 0.053 20.125
Postorbital length 0.319 20.469 20.679 0.379 20.272 20.030
Predorsal length 20.238 0.384 0.538 20.098 0.429 20.436
Dorsal length 0.573 0.448 0.109 0.573 0.265 20.098
Prepelvic length 0.468 0.482 20.200 0.431 0.521 0.323
Preanal length 20.090 0.574 20.283 0.135 0.533 0.447
Anal length 0.215 20.447 0.536 20.163 20.734 0.340
Caudal peduncle length 20.242 0.021 20.387 20.112 0.112 0.003
Caudal peduncle depth 0.623 20.354 0.210 0.420 20.712 0.277
Pectoral fin length 0.373 20.242 0.126 0.332 20.682 0.293
Pelvic fin length 0.381 0.793 0.051 0.251 0.371 0.633
Pectoral-pelvic fin distance 0.314 0.770 20.048 0.249 0.355 0.679
Pelvic-anal fin distance 0.729 20.029 0.217 0.792 20.039 20.167
Percent variance explained 32.433 14.159 8.928 31.952 14.421 8.215
TABLE 5. SUMMARY OF MERISTIC CHARACTER LOADINGS FOR PCA OF A. isfahanensis,A. sophiae,A. persicus,AND A.
vladykovi,AND PERCENT VARIANCE EXPLAINED BY EACH EIGENVECTOR. Total variance explained by the first three
eigenvectors is 68.94%. Heavy loadings (.75%) are indicated in bold.
Character
Eigenvector
123
Gillraker number 0.018 0.843 0.361
Lateral-line scales 0.638 20.238 20.412
Pectoral-fin rays 0.511 0.348 20.380
Pelvic-fin rays 0.512 0.427 20.326
Dorsal-fin rays 0.723 20.138 0.504
Anal-fin rays 0.807 20.198 0.314
Percent variance explained 35.054 18.820 15.066
252 COPEIA, 2006, NO. 2
similarity is probably the result of stabilizing
selection on morphological characters driven by
a common set of ecological/environmental vari-
ables. However, the species in these different
areas are not genetically interchangeable (Vill-
wock, 1964) and are genetically distinguishable
(Hrbek et al., 2002) in spite of extensive
morphological similarity. The species occupying
these disjunct areas are natural in that they
embody an evolutionary process, but do not
necessarily demonstrate the pattern in their
external morphology. It is important to remem-
ber that real evolutionary groups need not be
morphologically distinct, whereas morphological
categories are created as a direct function of
perceived distinction (Hey, 2001), and the two
are not the same.
Multivariate analyses of 20 morphometric
characters collected in males indicate that A.
isfahanensis differs significantly from A. sophiae
(Wilks’ Lambda F 520.711, d.f. 540, 60, P,
0.001), from A. persicus (Wilks’ Lambda F 5
18.670, d.f. 540, 350, P,0.001) and from A.
vladykovi (Wilks’ Lambda F 526.209, d.f. 540,
68, P,0.001). Same results are obtained for
morphometric comparisons of females: A. isfaha-
nensis differs significantly from A. sophiae (Wilks’
Lambda F 510.772, d.f. 540, 46, P,0.001),
from A. persicus (Wilks’ Lambda F 524.570, d.f.
540, 448, P,0.001), and from A. vladykovi
(Wilks’ Lambda F 512.184, d.f. 540, 28, P,
0.001). Multivariate analyses of six meristic
characters analyzed jointly for both sexes indicate
that A. isfahanensis differs significantly from A.
sophiae (Wilks’ Lambda F 5777.686, d.f. 512,
176, P,0.001), from A. persicus (Wilks’ Lambda
F52617.349, d.f. 512, 860, P,0.001), and
from A. vladykovi (Wilks’ Lambda F 51914.332,
d.f. 512, 166, P,0.001). For pair-wise
differences see Tables 1 and 2.
Although A. isfahanensis is statistically distin-
guishable in multivariate space as well as in
certain pair-wise comparisons from A. sophiae,A.
persicus, and/or A. vladykovi, with the exception
of lateral-line scale number in A. vladykovi, there
is no univariate or combination of univariate
characters that will unambiguously distinguish A.
isfahanensis from these other three Iranian
species. All morphometric and meristic charac-
ters overlap among species, and PCA analyses
show extensive overlap among species for both
meristic and morphometric characters (Fig. 3A,
B, C). The defining male color pattern of black
margins of the pelvic, anal, and dorsal fins is also
observed in Aphanius anatoliae from Turkey
(Wildekamp et al., 1999; Hrbek et al., 2002);
thus, this pattern is not exclusive to A. isfahanen-
sis. The female flank-bar pattern, which is similar
to that of A. persicus, is also found in females of A.
dispar and A. fasciatus. The only characters that
unambiguously distinguish A. isfahanensis from
all other species of Aphanius are 83 molecular
apomorphies in the 3303 mitochondrial DNA
characters analyzed in this study. Therefore
unambiguous species identification can only be
obtained through molecular analysis which in
principle is molecular bar-coding (Hebert et al.,
2003).
MATERIAL EXAMINED
Aphanius sophiae: NMC 79-0025, female,
31.6 mm SL, Iran, Fars, Kor River at Marv Dasht,
29u519N, 52u469300E; NMC 79-0059, 19 males,
18.1–39.3 mm SL, and 11 females, 24.5–31.9 mm
SL, Iran, Fars, Pulvar River, 8 km south of Sivand,
30u019300N, 52u579E, 18 April 1976, B. W. Coad
and S. Coad; NMC 79-0061, 3 females, 23.2–
32.1 mm SL, Iran, Fars, stream tributary to Pulvar
River, 14 km south of Sa’adatabad, 30u049N,
53u019E, 18 April 1976, B. W. Coad and S. Coad;
NMC 79-0062, 2 males, 24.0–26.6 mm SL, and 4
females, 26.5–42.0 mm SL, Iran, Fars, spring,
17 km south of Sa’adatabad, 30u059N, 53u009E,
18 April 1976, B. W. Coad and S. Coad; NMC 79-
0067, male, 25.7 mm SL, Iran, Fars, qanat at
Zarqan, 29u469N, 52u439E, 27 April 1976, B. W.
Coad and S. Coad; NMC 79-0292, 4 males, 26.5–
31.1 mm SL, and 2 females, 29.7–34.4 mm SL,
Iran, Fars, Lapu’i spring near Zarqan, 29u489N,
52u399E, 30 June 1974, H. Assadi; NMC 79-0342,
female, 39.4 mm SL, Iran, Fars, Kor River at
Band-e Amir, 29u499N, 52u519E, 22 Nov. 1977, B.
W. Coad; NMC 79-0498, 4 males, 19.7–25.2 mm
SL, and 2 females, 17.4–20.0 mm SL, Iran, Fars,
spring in Kor River basin, 30u059N, 52u279E, 4
Oct. 1978, B. W. Coad; CMNFi2004-0003 (Gen-
Bank AY593483, AY593492), 7 of 47, 20.7–
28.8 mm SL, Fars Province, Abolmahdi Spring,
17 km south of Sa’adat-shahr (Saadatabad),
30u069120N, 52u589380E, 14 April 2002, Y. Keivany
and S. Asadollah; CMNFi2004-0004 (GenBank
AY593482, AY593492), 8 of 38, 19.5–34.0 mm SL,
Fars Province, Malasskuh Spring, 29u529040N,
52u299200E, 15 April 2002, Y. Keivany and S.
Asadollah; uncatalogued (GenBank AY593481,
AY593490), 6, 25.7–37.0 mm SL, Fars Province,
Dolatabad Spring, 29u439050N, 52u509110E, 14
April 2002, Y. Keivany and S. Asadollah. Aphanius
persicus: CMNFi2004-0006 (GenBank AY593484,
AY593493), 15, 22.2–41.8 mm SL, Fars Province,
Nasrabad Spring near Imamzadeh Ibrahim,
29u359050N, 52u399080E, 15 April 2002, Y. Keivany
and S. Asadollah. Aphanius vladykovi: NMC 79-
0247, male, 36.6 mm SL, Iran, Shahrestan-e
Bakhtiari va Chahar Mahall, large pool, 3 km
HRBEK ET AL.—NEW APHANIUS FROM IRAN 253
west of Boldaji, 31u579N, 51u019E, 9 June 1977, B.
W. Coad and S. Mansoorabadi; NMC 79-0247A,
35 males, 21.6–36.4 mm SL, and 16 females,
23.5–40.2 mm SL, same locality as NMC 79-0247;
NMC 79-0248, male, 30.3 mm SL, Iran, Shahre-
stan-e Bakhtiari va Chahar Mahall, stream, 3 km
east of Boldaji, 31u559N, 51u059E, 9 June 1977, B.
W. Coad and S. Mansoorabadi; uncatalogued
(GenBank AY593486, AY593495), 4, 25.6–
37.0 mm SL, Chahar Mahall va Bakhtiari Prov-
ince, Ebrahimabad, 31u529300N, 51u109100E, 13
July 2002, S. Asadollah; uncatalogued (GenBank
AY593487, AY593496), 2, Chahar Mahall va
Bakhtiari Province, Taqanak, 32u129350N,
50u499290E, 26 March 2002, Y. Keivany and S.
Asadollah; uncatalogued (GenBank AY593485,
AY593494), 5, Chahar Mahall va Bakhtiari Prov-
ince, Madar-Dokhtar, 31u529120N, 51u089290E, 13
July 2002, Y. Keivany and S. Asadollah.
Additional molecular material included: Apha-
nius anatoliae splendens: MRAC 99-072-P-0040-
0041 (GenBank AF449287, AF449349), Lake
Salda, Turkey. Aphanius anatoliae anatoliae:
MRAC 96-053-P-0508-0517 (GenBank AF449292,
AF449354), Lake Eg˘irdir at Karaot, Turkey.
Aphanius anatoliae anatoliae: MRAC 97-033-P-
0172 (GenBank AF449293, AF449355), Lake
Isıklı near Beydilli, Turkey. Aphanius anatoliae
anatoliae: MRAC 92-136-P-0035-0043 (GenBank
AF449297, AF449359), Thaliye Canal by Sula-
masi, Turkey. Aphanius danfordii: MRAC A3-031-P-
0004-0009 (Genbank AF449299, AF449361), Soy-
salii springs in Sultan Swamps, Turkey. Aphanius
villwocki: MRAC A1-030-P-0001-0003 (GenBank
AF449303, AF449365), Pinarbas¸i in upper Sa-
karya River, Turkey. Aphanius asquamatus: unca-
talogued (GenBank AF449306, AF449368), Lake
Hazer, Turkey. Aphanius fasciatus: MRAC 92-136-
P-0078-0097 (GenBank AF449310, AF449372),
Lake Tuzla near Tarsus, Turkey. Aphanius sophiae:
uncatalogued (GenBank AF449314, AF449376),
Kor River basin near Shiraz, Iran. Aphanius
vladykovi: uncatalogued (GenBank AF449315,
AF449377), Boldaji basin, Iran.
ACKNOWLEDGMENTS
We thank the Iranian Environmental Protec-
tion Organization for permissions to collect
specimens. Specimens were collected in accor-
dance with established protocols of Isfahan
University of Technology. Support for this study
was provided by the U.S. National Science
Foundation postdoctoral grant INT-0002213
(TH), Isfahan University of Technology (YK),
and Canadian Museum of Nature (BWC). We
also thank S. Asadollah (Isfahan University of
Technology) for his help during field work, N.
Alfonso for assistance with morphometric and
meristic data collection and analysis, R. Wild-
ekamp for drawings of the types, and J. Cheverud
and Washington University Program in Popula-
tion, Ecology and Evolution for access to the
departmental sequencing facility.
LITERATURE CITED
ATCHLEY, W. R., C. T. GASKINS,AND D. ANDERSON.
1976. Statistical properties of ratios. I. Empirical
results. Syst. Zool. 25:137–148.
BENDER, F. 1968. Geologie von Jordanien. Borntrager,
Berlin-Stuttgart.
COAD, B. W. 1988. Aphanius vladykovi, a new species of
tooth-carp from the Zagros Mountains of Iran
(Osteichthyes: Cyprinodontidae). Environ. Biol.
Fishes 23:115–125.
———. 1996. Systematics of the tooth-carp genus
Aphanius Nardo, 1827 (Actinopterygii: Cyprino-
dontidae) in Fars Province, southern Iran. Biolo-
gia, Bratislava 51:163–172.
———. 2000. Distribution of Aphanius species in Iran.
J. Amer. Killifish Assoc. 33:183–191.
COYNE, J. A. 2004. Speciation. Sinauer Associates, Inc.,
Sunderland, Massachusetts.
DERCOURT, J., L. P. ZONENSHAIN,L.E.RICOU,V.G.
KAZMIN,X.LEPICHON,A.L.KNIPPER,C.GRAND-
JACQUET,I.M.SBORTSHIKOV,J.GEYSSANT,C.
LEPVRIER,D.H.PECHERSKY,J.BOULIN, J.-C. SIBUET,
L. A. SAVOSTIN,O.SOROKHTIN,M.WESTPHAL,M.L.
BAZHENOV,J.P.LAUER,B.BIJU-DUVAL,X.LE
PICHON,AND A. S. MONIN. 1986. Geological
evolution of the Tethys belt from the Atlantic to
the Pamirs since the Lias. Tectonophysics
123:241–315.
FULLER, R. C. 2002. Lighting environment predicts
the relative abundance of male colour morphs in
bluefin killifish (Lucania goodei) populations. Proc.
Roy. Soc. Lond. B 269:1457–1465.
GAUDANT, J. 1978. Sur une nouvelle espe`ce de
Poissons Te´le´oste´ens Cyprindondontiformes de
l’Oligocen de environs de manosque (Alpes de
haute-Provence). Ge´ ol. Me´dit. 5:281–290.
HEBERT, P. D. N., A. CYWINSKA,S.L.BALL,AND J. R.
DEWAARD. 2003. Biological identifications through
DNA barcodes. Proc. Roy. Soc. Lond. B
270:313–321.
HEY, J. 2001. Genes, Categories and Species: The
Evolutionary and Cognitive Causes of the Species
Problem. Oxford University Press, New York.
HRBEK, T., F. KU
¨C¸U
¨K,T.FRICKEY,K.N.STO
¨LTING,R.H.
WILDEKAMP,AND A. MEYER. 2002. Molecular phy-
logeny and historical biogeography of the Aphanius
(Pisces, Cyprinodontiformes) species complex of
central Anatolia, Turkey. Mol. Phylogenet. Evol.
25:125–137.
———, AND A. MEYER. 2003. Closing of the Tethys Sea
and the phylogeny of Eurasian killifishes (Cypri-
nodontiformes: Cyprinodontidae). J. Evol. Biol.
16:7–26.
———, K. N. STO
¨LTING,F.BARDAKC¸I,F.KU
¨C¸U
¨K,R.H.
WILDEKAMP,AND A. MEYER. 2004. Plate tectonics
and biogeographical patterns of the Pseudophoxinus
254 COPEIA, 2006, NO. 2
(Pisces: Cypriniformes) species complex of central
Anatolia, Turkey. Mol. Phylogenet. Evol.
32:297–308.
———, AND R. H. WILDEKAMP. 2003. Description of
a new Aphanius species (Pisces; Cyprinodonti-
formes) from the Sakarya River basin in the
western part of the central Anatolian plain, Turkey.
Ichthyol. Explor. Freshwaters 14:137–144.
KEIVANY, Y., AND N. SOOFIANI. 2004. Contribution to
the biology of Zagros tooth-carp, Aphanius vladykovi
(Cyprinodontidae) in central Iran. Environ. Biol.
Fishes 71:165–169.
KRIJGSMAN, W., F. J. HILGEN,I.RAFFI,F.J.SIERRO,AND
D. S. WILSON. 1999. Chronology, causes and pro-
gression of the Messinian salinity crisis. Nature
400:652–655.
LEVITON, A. E., R. H. GIBBS,JR., E. HEAL,AND C. E.
DAWSON. 1985. Standards in herpetology and
ichthyology: part I. Standard symbolic codes for
institutional resource collections in herpetology
and ichthyology. Copeia 1985:802–832.
MACEY, J. R., J. A. SCHULTE, II, N. B. ANANJEVA,A.
LARSON,N.RASTEGAR-POUYANI,S.M.SHAMMAKOV,
AND T. J. PAPENFUSS. 1998. Phylogenetic relation-
ships among agamid lizards of the Laudakia
caucasia species group: testing hypotheses of bio-
geographic fragmentation and an area cladogram
for the Iranian Plateau. Mol. Phylogenet. Evol.
10:118–131.
———, J. A. SCHULTE, II, H. G. KAMI,N.B.ANANJEVA,
A. LARSON,AND T. J. PAPENFUSS. 2000. Testing
hypotheses of vicariance in the agamid lizard
Laudakia caucasia from mountain ranges on the
northern Iranian Plateau. Ibid. 14:479–483.
POSADA, D., AND K. A. CRANDALL. 1998. MODELTEST:
testing the model of DNA substitution. Bioinfor-
matics 14:817–818.
RASTEGAR-POUYANI, N., AND G. NILSON. 2002. Taxon-
omy and biogeography of the Iranian species of
Laudakia (Sauria: Agamidae). Zool. Middle East
26:123–128.
REICHENBACHER, B., AND M. WEIDMANN. 1992. Fisch-
Odolithen aus der olligo-/mioza¨ nen Molaasse der
West-Schweitz und der Haut-Savoie (Frankreich).
Stuttgarter Beitr. Naturk., Ser. B 184:1–83.
RODRIGUEZ, R., J. L. OLIVER,A.MARIN,AND J. R.
MEDINA. 1990. The general stochastic model of
nucleotide substitution. J. Theor. Biol. 142:
485–501.
RONQUIST, F., AND J. P. HUELSENBECK. 2003. MrBayes
3: Bayesian phylogenetic inference under mixed
models. Bioinformatics 19:1572–1574.
SAUVAGE,H.E.1874.Noticesurlespossons
tertiares del’Auvergne. Bull. Soc. Hist. Nat. 8:
171–198.
SCHOLL, A., B. CORZILLIUS,AND W. VILLWOCK. 1978.
Beitrag zur Verwandtschaftsanalyse altweltlicher
Zahnkarpfen des Tribus Aphanii (Pisces: Cyprino-
dontidae) mit Hilfe elektrophoretischer Untersu-
chungsmethoden. J. Zool. Syst. Evol. Research
16:116–132.
SMITH, A. G., D. G. SMITH,AND B. M. FUNNELL. 1995.
Atlas of Mesozoic and Cenozoic Coastlines. Cam-
bridge University Press, New York.
SOKAL, R. R., AND F. J. ROHLF. 1994. Biometry: The
Principles and Practice of Statistics in Biological
Research, third edition. W. H. Freeman and
Company, New York.
VILLWOCK, W. 1964. Genetische Untersuchungen an
altweltlichen Zahnkarpfen der Tribus Aphaniini
(Pisces: Cyprinodontidae) nach Gesichtpunkten
der neuen Systematik. J. Zool. Syst. Evol. Research
2:267–382.
WEISROCK,D.W.,J.R.MACEY,I.H.UGURTAS,
A. LARSON,AND T. J. PAPENFUSS. 2001. Molecular
phylogenetics and historical biogeography
among salamandrids of the ‘‘true’’ salamander
clade: rapid branching of numerous highly di-
vergent lineages in Mertensiella luschani associated
with the rise of Anatolia. Mol. Phylogenet. Evol.
18:434–448.
WEST-EBERHARD,M.J.2003.Developmental
Plasticity and Evolution. Oxford University Press,
New York.
WILDEKAMP, R. H., F. KU
¨C¸U
¨K,M.U
¨NLU
¨SAYIN,AND W. V.
NEER. 1999. Species and subspecies of the genus
Aphanius Nardo 1897 (Pisces: Cyprinodontiformes)
in Turkey. Turk. J. Zool. 23:23–44.
(TH) UNIVERSITY OF PUERTO RICO–RIO PIEDRAS,
BIOLOGY DEPARTMENT,BOX 23360, UPR
STATION,SAN JUAN, PR, 00931, PUERTO RICO;
(YK) DEPARTMENT OF FISHERIES,FACULTY OF
NATURAL RESOURCES,ISFAHAN UNIVERSITY OF
TECHNOLOGY,ISFAHAN, 84156, IRAN;AND (BWC)
CANADIAN MUSEUM OF NATURE,P.O.BOX
3443, STATION D, OTTAWA, ON K1P 6P4,
CANADA. E-mail: (TH) hrbek@cnnet.upr.edu;
(YK) keivany@cc.iut.ac.ir; and (BWC) bcoad@
mus-nature.ca. Send reprint requests to TH.
Submitted: 26 March 2004. Accepted: 12 Dec.
2005. Section editor: D. Buth.
HRBEK ET AL.—NEW APHANIUS FROM IRAN 255
