2.1. Sequences of Catostomid GH
Partial to complete sequences of two distinct copies of GH were determined for 14 catostomid species; complete sequences for one of the GH copies were obtained from nine additional species (Table 1
). BLAST searches of the coding regions revealed high similarity of the new GH sequences with GH copies of Ictiobus bubalus
and other cypriniform fishes. The two GH copies are named GHI and GHII based on their sequence homology with GH copies in I. bubalus
We were able to produce complete coding region data for GHI for most catostomid species using methods described in the experimental section. We were able to produce data for the 5’ end of GHII (Exons 2 and 3) for most catostomid species using GHII specific primers developed in a previous study [16
]. However, despite several attempts involving a number of different techniques (also described in the experimental section), thus far we have only been able to produce data for the 3’ end of GHII for species representing tribes Erimyzonini and Catostomini of subfamily Catostominae, in addition to a previously published GHII sequence for I. bubalus
of subfamily Ictiobinae [ 16
The genomic organization of GH in suckers is the same as in other Cypriniformes [26
]. The complete GH genomic sequence comprises five exons and four introns with a total length of 1,500–2,700 nt depending on lengths of the four introns. Exons of different sucker species are of fixed lengths as follows: 10 (Exon 1), 140 (Exon 2), 117 (Exon 3), 162 (Exon 4), and 204 (Exon 5) nt. Introns vary in size across species, from 155–269 (Intron 1), 154–215 (Intron 2), 311–1,188 (Intron 3), and 102–154 (Intron 4) nt (Table 2
). The GHII genomic sequence is shorter than that of GHI, with much of the difference due to the substantially longer 3rd
intron of GHI.
The GH coding region of catostomids is 633 nt in length. The predicted amino acid (aa) sequences of GHI and GHII encode a protein of 210 aa, which is identical to the protein size reported for other cypriniforms [16
].The putative GH signal peptide cleavage site is serine at aa position 23, which gives a predicted mature polypeptide size of 188 aa, consistent with other cypriniform species [28
The two GH copies are very similar in both nt and aa sequence composition. Mean nt divergence between GHI and GHII is 9.61%. Mean pairwise aa sequence divergence between copies is 8.53%. Mean pairwise nt sequence divergence within paralogs (coding region data only) across catostomid species is 3.33% for GHI and 3.22% for GHII. Mean aa divergence within paralogs is 4.46% for GHI and 2.43% for GHII. The lower percentage in aa divergence for GHII is due to the incomplete data for several of the catostomine species.
An interesting and potentially evolutionarily significant difference in GH copies of suckers involves variation in the number of cysteine residues in the mature peptide. Pairs of cysteine residues form disulfide bonds, important to protein folding and stability [29
]. GH in all vertebrates has four cysteine residues in highly conserved positions in the amino acid sequence. Ostariophysan fishes have an unpaired, fifth cysteine in aa position 145. In GHI of catostomids, the extra cysteine is replaced by tyrosine. The functional significance of this disparity has yet to be established.
2.2. Phylogenetic Analysis of GHI and GHII
The consensus trees obtained with MP and ML analyses are identical. Only the MP tree is shown (Figure 1
). The MP analysis is based on 230 parsimony informative sites in the combined GHI/GHII data set (300 sites are constant). The MP consensus tree is 726 steps long. Order Cypriniformes (Node 1 in Figure 1
) is recovered as a monophyletic group with strong bootstrap support. Gyrinocheilus aymonieri
(Family Gyrinocheilidae) is strongly supported as the most basal cypriniform. Thus, GH data does not support a monophyletic Superfamily Cobitoidea inclusive of gyrinocheilids, loaches and catostomids, as supported by morphology [30
] and analysis of multiple nuclear genes and mitogenome data [24
Two strongly supported interfamilial groups make up the strongly supported sister group to Gyrinocheilus aymonieri. The first of these groups (Node 2) comprises a strongly supported family Cyprinidae (Node 3), sister to a strongly supported group of GHII sequences for representatives of tribe Catostomini (Node 4). The second group (Node 5) comprises a strongly supported basal group of cobitids and balitorids (Node 6), sister to a strongly supported group of GHI and GHII sequences representing other subfamilies and tribes of family Catostomidae (Node 7).
Family Cyprinidae comprises strongly supported subfamily groups of Cyprinines, and Leuciscines plus Gobionines. Within subfamily Cyprininae, the two copies of GH in tribe Cyprinini form a strongly supported monophyletic group, with sequences for each of the copies forming strongly supported monophyletic sister groups.
Five nt substitutions link cyprinid GH sequences with GHII sequences of suckers representing tribe Catostomini, thus rendering the two copies of GH in suckers, and catostomids as a whole, non monophyletic. In contrast, the GHI portion of the tree is well-resolved, monophyletic, and more or less consistent with hypotheses of catostomid relationships based on other data [32
]. GHI of Myxocyprinus asiaticus
is most basal. This species is sister to a strongly supported group comprising a monophyletic Catostominae GHI plus a strongly supported group of Cycleptus elongatus
plus a monophyletic subfamily Ictiobinae GHI, the latter group comprising a monophyletic Carpiodes
plus a monophyletic Ictiobus
. The catostomid GHI tree is the strongly supported sister group to GHII sequences for the remaining catostomid species. In the latter group, Ictiobus bubalus
GHII is basal and sister to a strongly supported group comprising GHII sequences for species representing tribes Erimyzonini, Moxostomatini and Thoburniini of subfamily Catostominae.
The sister group relationship of Catostomini GHII sequences with cyprinids was unexpected. Of the five nt substitutions inferred along this branch, four are not shared with other catostomid GH sequences, and two of these substitutions result in aa changes that are also not shared with other catostomids (valine to methionine at aa position 90 and leucine to methionine at aa position 169). The two aa substitutions are in C-terminal end of the protein, corresponding to Exons 4 and 5. GHII data from this end of the gene is available only for Minytrema melanops among Tribes Erimyzonini, Moxostomatini and Thoburniini. GHII sequences of Tribe Catostomini share nine nt characters with GHI and/or GHII sequences from other catostomids and would likely share more if GHII data were more complete. Two of the nine substitutions result in aa changes that are convergent with aa character states in other catostomid GH sequences (serine to cysteine in aa position 14 [signal peptide] and glycine to aspartic acid in aa position 81). It is possible that missing GHII data from the 3’ end of the gene, especially for other tribes of catostomines, would have supported a different tree topology.
When all catostomid GHII sequences are constrained to be monophyletic, the resulting tree is 11 steps longer than the MP consensus tree. When Catostomini GHII sequences are constrained to be the sister group of catostomid GHI plus the remaining GHII sequences, the resulting tree is only four steps longer than the MP consensus tree. Based on Templeton test results, neither constraint tree is significantly longer than the MP consensus tree (GHII monophyletic: Z = −1.9149, p = 0.0555; Catostomini GHII sister to remaining catostomid GHI and GHII sequences: Z= − 0.8944, p = 0.5034).
2.3. Selection Tests
We compared coding sequences of the mature GHI and GHII proteins of catostomids to gain insight into the possible evolutionary forces affecting the divergence of the two copies of the hormone. The comparison revealed a lower number of non-synonymous differences per non-synonymous site (dN
) relative to the number of synonymous differences per synonymous site (dS
= 0.003, Z-test of positive selection), indicating a paucity of amino acid replacement changes compared with neutral expectations. Thus, the null hypothesis of strict neutrality (dN
) can be rejected in favor of the alternative hypothesis of purifying selection (dN
) for all catostomid species. Purifying selection is also suggested for pairwise comparisons of GHI and GHII of the cyprinids Carassius auratus
and Cyprinus carpio
. There is no evidence for positive selection among the GH sequences tested. The slow rate of divergence of the GH coding region observed across suckers and other cypriniforms is not surprising considering the protein’s critical role in promoting growth and differentiation at distant target sites [34
] as well as its secondary functions in autocrine/paracrine regulation of cellular differentiation during embryonic development [35