Corresponding author: Hamid Reza Esmaeili (
Academic editor: K. Piller
The molecular status of nine species of the genus
Jouladeh Roudbar A, Eagderi S, Esmaeili HR, Coad BW, Bogutskaya N (2016) A molecular approach to the genus
The genus
Based on recent research, eleven species were considered to occur in Iranian inland waters. First,
Distribution and sampling sites of
A comparison of populations of different
After anesthesia, fishes were fixed in 5% formaldehyde and later stored in 70% ethanol. Counts and measurements follow Standard length total length multidimensional scaling
DNA was extracted from muscle tissue at the base of the dorsal fin using a Genomic DNA Purification Kit (#K0512, Thermo Scientific Corporation, Lithuania) following the manufacturer’s protocol. The COI gene was amplified using primers FishF1-(5'-TCAACCAACCACAAAGACATTGGCAC-3') and FishR1-(5'-TAGACTTCTGGGTGGCCAAAGAATCA-3'), designed by Polymerase chain reaction deoxynucleotide triphosphate
The haplotypes were compared to published
Sequenced were Iranian populations of
Details of the specimens used for molecular analysis.
Species | Accession No. | Sampling site | Latitude | Longitude | Basin/drainage |
---|---|---|---|---|---|
|
|
Damghan Spring |
|
|
Dasht-e Kavir |
|
|
Damghan Spring |
|
|
Dasht-e Kavir |
|
|
Damghan Spring |
|
|
Dasht-e Kavir |
|
Aras River |
|
|
Caspian Sea | |
|
Aras River |
|
|
Caspian Sea | |
|
Aras River |
|
|
Caspian Sea | |
|
Aras River |
|
|
Caspian Sea | |
|
Hari River |
|
|
Hari River | |
|
Hari River |
|
|
Hari River | |
|
Hari River |
|
|
Hari River | |
|
Bid Sorkh River |
|
|
Tigris River | |
|
Bid Sorkh River |
|
|
Tigris River | |
|
Bid Sorkh River |
|
|
Tigris River | |
|
Chardavol River |
|
|
Tigris River | |
|
Qareh Chai River |
|
|
Namak Lake | |
|
Qareh Chai River |
|
|
Namak Lake | |
|
|
Qareh Chai River |
|
|
Namak Lake |
|
Doab River |
|
|
Namak Lake | |
|
Doab River |
|
|
Namak Lake | |
|
Nam River |
|
|
Dasht-e Kavir | |
|
Nam River |
|
|
Dasht-e Kavir | |
|
Nam River |
|
|
Dasht-e Kavir | |
|
Nor Abad River |
|
|
Tigris River | |
|
Nor Abad River |
|
|
Tigris River | |
|
Nor Abad River |
|
|
Tigris River | |
|
Pulvar River |
|
|
Kor River | |
|
Pulvar River |
|
|
Kor River | |
|
Pulvar River |
|
|
Kor River | |
|
Ghadamgah Spring |
|
|
Kor River | |
|
Herat (Masih Spring) |
|
|
Sirjan | |
|
Tajan River |
|
|
Caspian Sea | |
|
Tajan River |
|
|
Caspian Sea | |
|
Tajan River |
|
|
Caspian Sea | |
|
Tajan River |
|
|
Caspian Sea | |
|
Emamzadeh Hashem (Safid River) |
|
|
Caspian Sea | |
|
Chalavand River |
|
|
Caspian Sea |
Screening for diagnostic nucleotide substitutions relative to
As appropriate outgroup to root the constructed phylogenetic hypothesis,
, standard length , lateral head length , Kimura 2-parameter
– Canadian Museum of Nature, Ottawa – Zoological Museum of Shiraz University, Collection of Biology Department, Shiraz
COI barcodes were generated for a total of 36 Bayesian Inference Maximum Likelihood
Bayesian analysis (based on
Diagnostic nucleotide substitutions found in the mtDNA COI barcode region of
Nucleotide position relative to |
||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
N | 5519 | 5529 | 5532 | 5535 | 5538 | 5598 | 5601 | 5631 | 5649 | 5667 | 5673 | 5679 | 5682 | 5691 | 5694 | 5700 | 5701 | 5707 | 5713 | 5722 | 5731 | 5734 | 5765 | |
|
4 | T | G | C | A | A | A |
|
T | A | G | T | T |
|
A | C | C | G |
|
G | A | A | A | C |
|
3 | T | G | C | A | A | A | A | T | A | G | T | T | A | A | C | C | G | A | G | A | A | A | C |
|
3 |
|
G |
|
A | A | A | A | T | A | G | T | T | A | A | C | C |
|
A | G |
|
|
A |
|
|
4 | T | G | C | A | A | A | A | T |
|
G | T | T | A | A | C | C | G | A | G | A | A | A | C |
|
8 | T | G | C | A |
|
A | A | T | A | G | T | T | A | A | C | C | G | A | G | A | A | A | C |
|
3 | T | G | C | A | A | A | A | T | A | G | T | T | A | A | C | C | G | A | G | A | A | A | C |
|
5 | T |
|
C | A | A | A | A |
|
A | G | T | T |
|
A |
|
C | G | A |
|
A | A |
|
C |
|
2 | T | G | C | A | A | A | A | T | A | G | T | T | A |
|
C | C | G | A | G |
|
A | A | C |
|
4 | T | G | C |
|
A |
|
A | T | A |
|
|
|
|
A | C |
|
G | A | G | A | A | A | C |
N | 5776 | 5786 | 5789 | 5800 | 5815 | 5818 | 5821 | 5836 | 5854 | 5857 | 5885 | 5902 | 5911 | 5914 | 5920 | 5959 | 5965 | 5992 | 6001 | 6004 | 6019 | 6050 | 6092 | |
|
4 | G | C | C | T | T | A | C | A |
|
A | G | A | A | A |
|
T | T | T | C | C | G | T | C |
|
3 | G | C | C | T | T | G | C | A | A | A | G | A | A | A | A | T | T | T | T | C | G | T | C |
|
3 | G |
|
|
T | T |
|
|
A | A |
|
G | A |
|
|
A | T |
|
|
|
|
|
|
C |
|
4 | G | C | C | T | T | A | C | A | A | A | G | A | A | A | A | T | T | T | C | C | G | T | C |
|
8 | G | C | C | T | T | A | C | A | A | A | G | A | A | A | A |
|
T | T | T | C | G | T | C |
|
3 | G | C | C | T | T | A | C | A | A | A |
|
|
A | A | A | T | T | T | T | C | G | T | C |
|
5 | G | C | C | T | T | G | C |
|
A | A | G | A | A | A | A | T | T | T | C | C | G | T |
|
|
2 | G | C | C | T |
|
A | C | A | A | A | G | A | A | A | A | T | T | T | T | C | G | T | C |
|
4 |
|
C | C |
|
T | A | C | A | A | A | G | A | A | A | A | T | T | T | T | C | G | T | C |
Estimates of the average evolutionary divergence between Iranian
No. | Species | N | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 |
---|---|---|---|---|---|---|---|---|---|---|
1 |
|
4 | ||||||||
2 |
|
3 | 3.08 | |||||||
3 |
|
3 | 6.78 | 5.75 | ||||||
4 |
|
4 | 2.76 | 1.08 | 5.70 | |||||
5 |
|
8 | 3.74 | 0.97 | 6.18 | 1.72 | ||||
6 |
|
3 | 3.08 | 1.04 | 5.94 | 0.73 | 1.68 | |||
7 |
|
5 | 2.90 | 3.57 | 7.12 | 3.62 | 4.61 | 3.94 | ||
8 |
|
2 | 4.21 | 2.54 | 6.41 | 2.58 | 3.19 | 2.54 | 5.52 | |
9 |
|
4 | 5.09 | 3.17 | 7.76 | 3.21 | 3.83 | 3.17 | 5.99 | 3.64 |
Cheshmeh Ali (Ali Spring), Damghan River tributary, Iran.
The eye is small, its horizontal diameter enters interorbital width 1.2 times. The snout is short and stout, its length only slightly exceeds the eye diameter. The upper jaw slightly projects over the lower jaw. The mouth is small, terminal, the mouth cleft is slightly curved, and the tip of the mouth cleft is on a level with the lower margin of the pupil. The posterior end of the lower jaw is on a vertical with the anterior margin of the pupil. The body depth enters
Dorsal-fin rays are 4 unbranched and 8½ branched, anal fin rays are 3 unbranched and 12½ branched, pectoral-fin branched rays are 13, and pelvic-fin branched rays are 7. The anal-fin origin is on a vertical from the posterior end of the dorsal-fin base. Total lateral-line scales number 46 and those to posterior margin of hypurals 44, scales around caudal peduncle 17, scales above lateral line to dorsal fin origin are 9, scales below lateral line to anal-fin origin are 4, scales below lateral line to pelvic-fin origin are 4, and midline predorsal scales are 27. Pharyngeal teeth 2.5-4.2. Gill rakers number 6, they are short and stubby, the longest touching the adjacent one when appressed. Total vertebrae number 40 (abdominal vertebrae 20, caudal vertebrae 20). Predorsal vertebrae number 13.
The peritoneum is silvery with fine melanophores. The lateral line is clearly delineated by darker pigment above and below, but this is obscured on the caudal peduncle by the flank stripe. Some pigment on flank scales above the lateral line give the impression of stripes. A mid-flank stripe is evident, darkest on the caudal peduncle. The back and top of the head are dark, the belly is light with almost no melanophores. Melanophores are dense dorsally on the flank becoming progressively less ventrally. All fins have melanophores lining the rays, and the dorsal, anal and caudal fins have melanophores on the membranes, with very few melanophores on the pectoral- and pelvic-fin membranes. The unbranched pectoral-fin ray is lined with melanophores on its inner margin.
Live specimen of
Morphometric data for the holotype of
Character | Holotype | Min | Max | Mean | SD |
---|---|---|---|---|---|
67.0 | 54.6 | 84.4 | |||
Body depth at dorsal-fin origin (% |
30.9 | 28.9 | 33.3 | 31.14 | 1.16 |
Depth of caudal peduncle (% |
12.9 | 12.0 | 14.1 | 13.01 | 0.51 |
Depth of caudal peduncle (% length of caudal peduncle) | 60.6 | 57.3 | 68.1 | 63.10 | 2.91 |
Body width at dorsal-fin origin (% |
12.5 | 12.3 | 15.9 | 14.32 | 1.01 |
Caudal peduncle width (% |
4.6 | 3.9 | 5.6 | 4.66 | 0.42 |
Predorsal length (% |
54.9 | 53.0 | 57.1 | 55.12 | 1.23 |
Postdorsal length (% |
35.4 | 33.2 | 40.2 | 35.34 | 1.64 |
Prepelvic length (% |
49.1 | 45.9 | 53.2 | 49.15 | 1.44 |
Preanal length (% |
66.3 | 62.9 | 69.7 | 66.38 | 1.52 |
Pectoral – pelvic-fin origin length (% |
23.6 | 21.4 | 27.2 | 23.85 | 1.49 |
Pelvic – anal-fin origin length (% |
19.6 | 16.6 | 20.6 | 18.37 | 1.11 |
Length of caudal peduncle (% |
21.4 | 19.0 | 22.4 | 20.64 | 0.85 |
Dorsal-fin base length (% |
14.0 | 11.6 | 19.7 | 13.71 | 1.57 |
Dorsal fin depth (% |
22.5 | 18.3 | 23.9 | 20.93 | 1.29 |
Anal-fin base length (% |
17.1 | 14.7 | 19.5 | 17.45 | 1.42 |
Anal fin depth (% |
15.0 | 12.3 | 15.2 | 13.74 | 0.90 |
Pectoral-fin length (% |
19.9 | 17.7 | 21.5 | 19.66 | 1.00 |
Pelvic-fin length (% |
17.1 | 13.3 | 18.7 | 16.39 | 1.17 |
Head length (% |
27.2 | 24.5 | 28.1 | 26.74 | 0.88 |
Head length (% body depth) | 87.9 | 77.6 | 92.4 | 85.96 | 3.54 |
Head depth at nape (% |
21.1 | 19.0 | 22.5 | 21.07 | 0.96 |
Head depth at nape (% |
77.8 | 73.6 | 83.7 | 78.83 | 2.79 |
Head depth through eye (% |
54.9 | 52.5 | 66.4 | 57.71 | 3.16 |
Maximum head width (% |
13.3 | 12.2 | 14.9 | 13.68 | 0.61 |
Maximum head width (% |
49.0 | 48.4 | 56.5 | 51.21 | 2.27 |
Snout length (% |
7.8 | 6.5 | 7.9 | 7.33 | 0.35 |
Snout length (% |
28.9 | 24.4 | 29.3 | 27.42 | 1.11 |
Eye horizontal diameter (% |
6.9 | 6.5 | 7.9 | 7.04 | 0.38 |
Eye horizontal diameter (% |
25.5 | 23.5 | 28.2 | 26.35 | 1.36 |
Eye horizontal diameter (% interorbital width) | 81.6 | 71.3 | 87.8 | 78.22 | 4.40 |
Postorbital distance (% |
47.8 | 47.8 | 53.6 | 50.81 | 1.68 |
Interorbital width (% |
8.5 | 7.8 | 9.7 | 9.02 | 0.49 |
Interorbital width (% |
31.3 | 31.3 | 36.2 | 33.72 | 1.44 |
Length of upper jaw (% |
28.1 | 28.1 | 35.3 | 31.81 | 1.65 |
Length of upper jaw (% |
7.6 | 7.5 | 9.8 | 8.51 | 0.54 |
Length of lower jaw (% |
11.2 | 9.7 | 12.4 | 10.99 | 0.64 |
Length of lower jaw (% |
41.2 | 37.4 | 44.6 | 41.10 | 1.70 |
Length of lower jaw (% interorbital width) | 131.6 | 109.8 | 142.8 | 122.09 | 7.29 |
Length of lower jaw (% depth of operculum) | 94.3 | 90.7 | 104.3 | 96.87 | 4.31 |
Depth of operculum (% |
43.7 | 38.5 | 46.3 | 42.47 | 1.82 |
|
|||||
Interorbital width/eye horizontal diameter | 1.2 | 1.1 | 1.4 | 1.28 | 0.07 |
Snout length/eye horizontal diameter | 1.1 | 1.0 | 1.1 | 1.04 | 0.05 |
Head depth at nape/eye horizontal diameter | 3.0 | 2.8 | 3.2 | 3.00 | 0.13 |
Head length/caudal peduncle depth | 2.1 | 1.9 | 2.3 | 2.06 | 0.08 |
Length of caudal peduncle/caudal peduncle depth | 1.6 | 1.5 | 1.7 | 1.59 | 0.07 |
Length of lower jaw/caudal peduncle depth | 0.9 | 0.8 | 1.0 | 0.85 | 0.05 |
Pectoral-fin length/pectoral – pelvic-fin origin distance | 0.8 | 0.7 | 1.0 | 0.83 | 0.08 |
Predorsal length/head length | 2.0 | 1.9 | 2.2 | 2.06 | 0.07 |
In 24 paratypes (
In 39 paratypes (
Mature males bear tubercles on the unbranched and branched fin rays, in a single row branching into two distally on the branched rays. These are most prominent on the pectoral, pelvic and anal fins. Tubercles line scale margins in a single row of up to six tubercles, in particular over the anal fin and on the lower caudal peduncle. Scales below the dorsal fin are also lined with tubercles but to a much lesser extent than those above the anal fin. Flank scales generally may bear tubercles but many do not and anterior flank scales may have only a single tubercle. Minute tubercles are present on the dorsal and upper head surface.
Pigmentation consists of a darker back fading to a silvery white belly, three to four rows of large dark spots above lateral line starting from posterior part of operculum to posterior level of anal fin, continuing with two rows behind anal fin to base of caudal fin, small black spots on the operculum, behind and below the eye, smaller and less dark spots between the eye and upper jaw, a lateral line demarcated by pigment above and below it (the typical “stitched” pattern in many
The species name links to the type locality, Damghan (Cheshmeh Ali, Damghan River tributary). Proposed common name: Damghan riffle minnow, Mahi-e-Khayateh-e-Damghan (Farsi).
Two views of Cheshmeh Ali, Damghan, type locality of
Results of a DMS analysis showing observed similarities/dissimilarities (distances) between the examined groups of samples of
Uncatalogued
Mean values of some meristic characters of
Species | Branched anal-fin rays (without «½») | Branched dorsal-fin rays (without «½») | Gill rakers | Lateral-line scales to margin of hypurals | Total vertebrae | Predorsal vertebrae | Abdominal vertebrae | Caudal vertebrae | Difference between abdominal and caudal vertebral counts | |
---|---|---|---|---|---|---|---|---|---|---|
|
12.16 | 7.95 | 7.37 | 48.87 | 41.25 | 13.65 | 20.72 | 20.53 | 0.19 | |
|
13.16 | 8.00 | 7.95 | 48.50 | 40.57 | 13.18 | 20.13 | 20.41 | -0.25 | |
|
12.87 | 8.00 | 8.62 | 48.96 | 40.26 | 12.63 | 19.89 | 20.37 | -0.48 | |
|
12.82 | 7.95 | 8.58 | 49.02 | 40.27 | 12.18 | 19.73 | 20.55 | -0.82 | |
|
13.11 | 7.86 | 8.14 | 49.12 | 40.29 | 12.66 | 20.09 | 20.26 | -0.14 | |
|
12.38 | 7.89 | 7.62 | 48.64 | 40.24 | 12.30 | 20.08 | 20.22 | -0.14 | |
|
14.72 | 8.23 | 7.22 | 48.81 | 41.05 | 13.21 | 19.89 | 21.16 | -1.21 | |
|
12.53 | 7.90 | 6.70 | 45.10 | 39.93 | 12.24 | 19.78 | 20.15 | -0.36 | |
|
13.43 | 8.00 | 6.50 | 45.40 | 40.90 | 12.93 | 19.77 | 21.17 | -1.40 | |
|
11.77 | 7.88 | 6.88 | 42.65 | 40.08 | 13.08 | 20.18 | 19.90 | 0.28 | |
|
11.83 | 8.11 | 7.00 | 46.12 | 39.72 | 12.22 | 19.80 | 19.90 | -0.10 | |
|
12.38 | 7.92 | 8.54 | 48.88 | 39.88 | 13.26 | 19.76 | 19.94 | -0.18 | |
|
9.30 | 7.30 | 7.22 | 45.62 | 40.53 | 13.44 | 21.00 | 19.54 | 1.44 |
When compared to
The new species differs from
As can be seen from Fig.
Extensive comparative material is listed in
The present data comprise the first comprehensive molecular study based on the COI barcode region on the genus
Lineage I includes two species,
Lineage II comprises highly diverse
Lineage III comprises one monotypic undescribed species (accession number:
Lineage IV is formed by the highly diverse
Lineage IV,
To conclude, the genetic analyses supported the validity of many morphologically distinguishable species of the genus
We express our sincere thanks to G. Sayyadzadeh for her kind help in fish collection and laboratory analysis, A. Gholamifard, A. Gholamhosseini, R. Zamanianjejad, S. Ghasemian, S. Mirghiasi, and B. Parsi for helping with fish collection, and the Environment Departments of Semnan, Fars, Markazi, Qom, and Ardabil provinces for their kind cooperation in visiting the collection sites. We are grateful to O.A. Diripasko (Institute of Fisheries and Marine Ecology, Ukraine) for his valuable assistance with the statistical analyses. The research work was funded by Shiraz University (approved by the Ethics Committee of the Biology Department, ECSU-909789), Tehran University, and the Canadian Museum of Nature. We also thank M. Geiger and J. Freyhof from the FREDIE project.