Send Orders for Reprints to Reprints@benthamscience.net Carduelini New Sistematics: Crimson-winged Finch (rhodopechys Sanguineus) Is Included in " Arid-zone " Carduelini Finches by Mitochon- Drial Dna Phylogeny

Rhodopechys sangineus phylogeny together with a group of Carduelini finches has been analyzed. Mitochon-drial cyt b molecule has been used for species comparison and maximum likelihood and Bayesian methods have been employed in order to obtain a solid phylogeny. Compared Carduelini finches groups include: Greenfinches, " Arid-Zone " finches and Genera Rhodopechys and Pyrrhula species. Our results lead to conclude: 1) Genus Rhodopechys included species should need a new taxonomic classification; 2) Genus Pyrrhula shares a common ancestor with " Arid-Zone " finches group; the latter is phylogenetically a separate clade, including species from Carpodacus, Rhodopechys and Leu-costicte Genera, and 3) Pinicola enucleator belongs to Genus Pyrrhula and seems to be ancestral. Results show that a sis-tematics revision of Carduelini tribe bird species is required.


INTRODUCTION
Songbirds evolution (Aves, Passeriformes, Passeri) have frequently been studied.Their phenotypic and molecular evolutions are not always concordant for all cases [1,2].Thus, the similarities in morphology and behavior may originate shared features among genetically unrelated species occurring in similar environments; conversely, different features may be present among genetic sister taxa thriving under different environments.This phenotypic plasticity has already been described for other bird species [3,4].Thus, some genetic and/or phenotypic traits may not correlate with the evolutionary histories of the birds.On the other hand, mitochondrial cytochrome b gene (cyt-b) sequencing has been widely used for molecular systematics studies.Cyt-b gen has been proved to be helpful for defining evolutionary relationships among distant or closely related taxa [5,6].
Rhodopechys sanguineus thrives discontinuously across all desert strip margins and mountains that go from Africa to China.Its presence in Atlas Mountains may be due to winter migration, but it has been recorded in breeding season [7].It seems that this species is mostly an altitudinal migrant although in China it moves from breeding area in East China range to North Central China in winter.It is found in Lebanon, Mount Hermon (Israel) and probably Syria; strikingly it is also found in Caucasus Mountains, North Ossetia and Kashmir-Indian area (Chitral and Ladakh) [7].
Birds included within the Carduelini tribe (Genera Rhodopechys, Carpodacus and Leucosticte) have been shown to belong to the same evolutionary radiation by using molecular phylogenetic analyses [8].Our phylogenetic analyses based cytochrome b gene (cyt-b) nucleotide sequences show that some of these birds (Rhodopechys mongolica, Rhodopechys githaginea and Carpodacus nipalensis) do not go in dendrograms with their respective phenetically defined allies and/or classical Genus classification [7].This newly defined group of birds lives in both hot and cold arid areas and are phenetically distinct, probably because of their adaptation to diverse extreme environments.Both maximum likelihood and Bayesian inference dendrograms support the existence of this new group which seems to have appeared about 14 million years ago [8].
In the present work, we study the phylogeny of Crimsonwinged Finch (Rhodopechys sanguineus) in order to complete the split family and new classification of Rhodopechys and "Arid-Zone" finches included species [8,9,10].Our suggested taxonomic revisions are detailed at the end of Discussion section.

Bird Samples, DNA Extraction and PCR Amplifications
Sixty two species of songbirds (order Passeriformes, suborder Passeri) have been included for study (see Table 1).They belong to the tribe Carduelini.Rhodopechys sanguineus was DNA-sequenced (four differents birds) and analyzed in the present work together with relevant Carduelini species.Other sequences used in this analysis were retrieved from the GenBank (see Table 1) [8,9,[11][12][13].DNA extraction was done using a standard protocol [14].Amplification and sequencing of cyt b gene 924 base pairs (bp) was performed as previously described [9].

Phylogenetic Analyses
Sequence was analyzed for stop codons with MEGA v. 3.1 as described [15].Phylogenetic dendrograms were obtained using maximum likelihood (ML) [16] in PAUP* v. 4.0b10 [17] and Bayesian (BI) [18,19].Model test v. 3.7 [20] was used to find out a DNA substitution model that fits the data best.Best model was then used in both the ML and BI analyses.Linearized ML dendrograms were obtained with PAUP* v. 4.0b10 [17] with the estimated branch length according [21] which assumes that the rates among the evolutionary lineages may not be constant.Tree calculation strategy consisted of a heuristic search with NNI (Nearing-Neighbor Interchange) swapping algorithm.Robustness of nodes was assessed by 1000 bootstrap replicates in the ML analyses.The parameters rates defining the model of evolution were allowed to change in the BI analysis after each generation in order to increase the likelihood of resulting trees.Therefore, none of the parameters were a priori fixed.
In BI analyses, two independent runs (with one cold and three heated chains each) were performed along with 5 million generations.Trees were sampled every 100 generations and the first 12,500 samples were discarded as 'burn-in'.Split frequencies average standard deviation approached to zero being around 0.01 at the end of the analysis.Posterior probability values (ppv) indicate the robustness of the nodes in the BI.Phylogenetic analyses the lark-like bunting, and chaffinch, Fringilla coelebs (family Fringillidae, subfamily Fringillinae), was used as an outgroup (Table 1).Phylogenetic analyses using ML and BI methodology gave on a congruent topology (Figs. 2, 3).Several monophyletic clades were found, i.e.: crossbills (Genus Loxia; ML bootstrap=100 (Fig. 2), BI ppv=1.00 (Fig. 3)) and bullfinches (Genus Pyrrhula; ML bootstrap=83 (Fig. 2), BI ppv=1.00 (Fig. 3)).Rosefinches (Genus Carpodacus) form a polyphyletic clade, as it was already shown [11].Genera Carduelis and Serinus species, however, are clustered in several paraphyletic groups as already described [13].Also, some monotypic Genera group with another different Genus: Pinicola enucleator (pine grosbeak) forms a clade with Genus Pyrrhula (ML bootstrap=81 (Fig. 2), BI ppv=0.91 (Fig. 3)), Uragus sibiricus (long-tailed rosefinch) goes with Carpodacus rubicilloides (ML bootstrap= 30 (Fig. 2), BI ppv=0.92 (Fig. 3)) and Haematospiza sipahi (scarlet finch) is clustered together with Carpodacus erythrinus (ML bootstrap=49 (Fig. 2), BI ppv=0.97 (Fig. 3)); this is expected from our previous work [11].R. obsoleta is grouped with Greenfinches (Carduelis), splitting Genus Rhodopechys, as expected [9].Citril finch (Carduelis citrinella) groups with the European goldfinch (Carduelis carduelis), which is congruent with previous reports [9,22].A phylogenetic group consistently contained two species of Genus Rhodopechys (R. mongolica and R. githaginea), two subspecies of Leucosticte arctoa (L.a. arcota and L. a. tephrocotis) and the dark-breasted rosefinch (Carpodacus nipalensis).This group has a bootstrap value of 60 in the ML dendrogram (Fig. 2) and a posterior probability value of 1.00 in the Bayesian tree (Fig. 2).This new group might represent a Carduelini group of "Arid-Zone" birds.We expected that Rhodopehcys sanguineus, Leucosticte brandti, and Leucosticte nemoricola may probably belong to this group [7].Samples and further analyses are necessary to assess this hypothesis.However, it is clear from present paper results that Rhodopechys sanguineus is in fact included in within "Arid-Zone" finches group (Fig. 2, 3), probably being one of the most ancient extant bird of these species.Considering the linearized maximum likelihood genetic timing and the corresponding cladogenesis divergence among the taxa analyzed (see Fig. 2 footnote), Genera Serinus and Carduelis diverged more recently and probably in a simultaneous fashion.Rosefinches seem to have diverged earlier; and the bullfinches (Pyrrhula) as well as the "Arid-Zone" finches diverged even earlier at the base of the tree (Fig. 2).

Eurasian thriving range of
Bayesian phylogenetic trees are fully concordant with our maximum likelihood results (Figs. 2, 3; not shown).

DISCUSSION
Some topics and questions arise from our results.

Miocene Climate Change
The Miocene Epoch had a initial cold phase (Mi-1) followed by a general Earth warming; tundra was substituted by conifer woods in the corresponding areas between 17 and 14.5 million years ago (MYA) [23]; temperatures by then were 6ºC higher than at present [23].However, after about 10 MYA temperatures dropped and finally Antarctica and Greenland became glaciated about 6 MYA.Together with this general cooling, vast arid regions were established in Asia and Africa [23].In addition, Tibet plateau underwent its higher uplift during this cold Miocene period [23,24].Then a heavy rain regime in the high Tibetan-Himalayan Mountains occurred that gave birth to the deep and big rivers (Ganges, Bramaputra, Yangtze and Mekong), which carry 25% of the total suspension materials that reach Oceans on Earth.Ancestors of "Arid-Zone" Carduelini finches might have appeared 13.5 MYA together with Genus Pyrrhula (Bullfinches) witch seems a sister Genera which includes Pinicola enucleator.Bullfinches would have occupied a more humid and full of vegetation habitat than "Arid-Zone" finches (Table 1) [7].

Rhodopechys Sanguineus and Genus Rhodopechys Split
Rhodopechys sanguineus has been included within "Arid-Zone" finches together with Genus Leucosticte and Carpodacus nipalensis.They all share one phenotypic characteristic: a different degree of reddish-pink plumage.Also, all of them thrive in extreme climates over the World; habitats are correspondingly desert margins or mountains with scarce vegetation.Rhodopechys sanguineus seems to be ancestral to Rhodopechys githaginea (Trumpeter Finch) and Rhodopechys mongolica (Mongolian Finch); It appeared on Earth about 10 million years ago according to our linearized ML and Bayesian dendrograms results (Figs. 2 and 3).

Genus Pyrrhula and "Arid-Zone" Finches
Ancestors of the novel group of "Arid Zone" Carduelini finches might have appeared 13.5 MYA together with Genus Pyrrhula (Bullfinches) (Fig. 2) which seems a sister Genus.Bullfinches would have occupied a more humid and full of vegetation habitat than most "Arid-Zone" finches (Table 1) [7]; see below, more coloured Leucosticte finches and C. nipalensis.Also, all Pyrrhula species show a Paleartic distribution, including their extant ancestor Pinicola enucleator (Pine Grosbeak), which should be included within Genus Pyrrhula [11].

Rhodopechys
Pine Grosbeak (P.enucleator) seems to be the one Pyrrhula species found in North America in addition to show a Holoartic distribution it seems to be Genus Pyrrhula ancestor [11] (Figs. 2, 3) [7].Coloured Bullfinch species are concordant with its wood bud-containing inhabitat.This phylogeography information is roughly coincidental with the appearance of wide arid areas in Africa and Asia."Arid-Zone" finches evolutionary radiation might have started around this time most likely in Asia (or in Africa) from where some of the lineages might have undergone dispersal during warmer conditions.The most ancient extant ancestor is Rhodopechys sanguineus (Fig. 2, 3).The "Arid-Zone" finches feather colours are not homogeneous and some Leucosticte and Carpodacus finches included in the group tend to bear more melanine pigment.This may suggest a more recent and separate change in the "Arid-Zone" finches environments.Habitats which are more humid (i.e., those of Leucosticte and Carpodacus nipalensis in comparison with Rhodopechys habitats) favour melanine dyed feathers, because it protects plumage from bacterial degradation by humidity [25].Carotenoids, the other major pigments responsible for plumage colouration [26], are not available for bird feeding in dry areas, but Carpodacus nipalensis also feeds on berries at high altitudes [7] and this may enhance its intense male purple face colouring at breeding.Also, C. nipalensis has the darkest plumage colours among rosefinches [7], resembling Leucosticte finches in this respect.In summary, according to our phylogenetic analyses based on mitochondrial cytochrome b DNA sequences, the Genera Rhodopechys and Carpodacus should be revised because this "Arid-Zone" finches seems to be a separate evolutionary group that comprises species belonging to both of the previously admited Genera.Also, Genus Pyrrhula should be regarded as sharing its extinct (or non-analyzed) ancestor with "Arid-Zone" finches (see Fig. 2).Further analyses using other genetic markers could be required to complete these phylogenetic findings, and a taxonomical revision of these Genera (Rhodopechys, Pyrrhula and "Arid-Zone" finches) and Carduelini tribe is needed since only monophyletic clades should be used in a phylogenetical systematics [1,11].Thus, our suggested conclusions of present and previous studies [8,9,11] are: 1.The polytomies newly found in our Carduelini finches phylogenetic dendrograms (Figs. 2, 3) indicate that a systematics revision of these groups is required [8,9,11].

Fig. ( 1 ).
Fig. (1).Geographic distribution and photographs of present paper studied species.Leucosticte arctoa (red) thrives from Central Asia (Altai) to eastern Siberia, Japan, Alaska to western Canada and western USA; Rhodopechys sanguineus (orange) thrives in Morocco, central Turkey and Middle East to northwest China mountains; Rhodopechys githaginea (yellow) thrives in Canary Islans North África, Middle East to Central Asia and northern mountain; Rhodopechys mongolica (green) is distributed in Eastern Turkey to Central Asia, Kashmir, western and northern China; Carpodacus nipalensis (blue) inhabit in Himalayas from northern lndia to southern Tibet and western China [7].