Subspecies identification of Lesser Whitethroats, Sylvia curruca, wintering in southern Iran as inferred from DNA sequences (Aves: Sylviidae)

The Lesser Whitethroat Sylvia curruca complex has a wide distribution in the east and west Palaearctic and comprises phenotypically very similar, but genetically distinct forms. In order to determine the origin of the birds wintering on Qeshm Island in the Persian Gulf, we studied the nucleotide sequences of the mitochondrial cytochrome b gene of 23 individuals and compared them with sequences publicly available on Gen-Bank. The birds from Qeshm were attributed to the subspecies S. c. halimodendri, probably breeding in areas of Kazakhstan and China, and formed two distinct clades. As the genetic distance between these groups is very low, it is likely that they represent different populations of the same taxon.


Introduction
The distribution of the Lesser Whitethroat Sylvia curruca extends from the west of the Palaearctic to the east of Russia, and includes large parts of the Tibetan Plateau along with parts of India (Shirihai et al., 2001). One of its southernmost distribution areas is Qeshm Island and Lut Desert in southern Iran (Olsson et al., 2013). Several subspecies have been recognized: Sylvia c. curruca, S. c. althaea, S. c. minula, S. c. blythi, S. c. halimodendri, S. c. margelanica, and S . c. zagrossiensis (Abdilzadeh et al., 2020;Olsson et al., 2013). Subspecies of Sylvia curruca are phenotypically very similar and often misidentified (Dement'ev & Gladkov, 1968;Loskot, 2005;Olsson et al., 2013;Svensson, 1992), but are clearly distinct genetically. In a comprehensive review, Olsson et al. (2013), using gene sequences of 213 individuals from the entire distribution range of this species, identified six subspecies that were placed in distinct clades. In Iran, Abdilzadeh et al. (2020), using the mitochondrial marker cytochrome b gene (cytb), identified three clades in different regions of the country, which were assigned to S. c. althaea, S. c. halimodendri and S. c. curruca. However, there are still ambiguities in the taxonomy of the species regarding the southern part of the country, particularly in the genetic identity of birds wintering there. We therefore examined DNA sequences of a series of birds wintering in the Persian Gulf.

Material and Methods
The study area includes three different localities on Qeshm Island, an about 1491 km 2 large island in the Persian Gulf and situated at the southern range of the Lesser Whitethroat ( Figure 1). Twenty-three Lesser Whitethroats were caught with mist nets between 9 and 21 December 2019, with the permission of the Environmental Protection Organization. Additionally, 29 DNA publicly available sequences of known subspecies of Sylvia curruca were downloaded from GenBank (Table S2). GenBank accession number and geographical coordinates of sampling points are given in Table S1. One microliter of blood sample was taken from the brachial wing vein (or cutaneous ulnar vein) of each individual using an insulin syringe (Kelly & Alworth, 2013). In addition, a pair of secondary feathers were symmetrically picked up from the wings, and the bird was returned to nature and to the same place. After the blood sampling, blood samples were transferred to the laboratory in a queen buffer at 4°C temperature. After the feathers were dried in the open air, they were placed in sterile nylons to prevent fungal infection and were transported to the laboratory along with the blood sample for molecular testing (Segelbacher, 2002).
The phenol-chloroform method was used to extract DNA from samples (McKiernan & Danielson, 2017;Ong & Vellayan, 2008). In this method, out of 50 μl of 10% SDS solution, 500 μl of STE buffer (sodium chloride, tris, and EDTA), and 5 μl of proteinase k, Phenol-chloroform isoamyl (500 μl) at a ratio of 25:24:1, 400 μl of absolutely cold ethanol, 75 μl of 75% ethanol and 45 μl of distilled water were used. Then the obtained DNA solution was stored in the refrigerator and then at -20°C. In order to check the quantity and quality of the extracted DNA, a nanodrop device (Thermo 2000C model) was used. The polymerase chain reaction was then used to amplify specific DNA fragments (Table S3). The primers L14841 and H15149 were used for the amplification of the cytb fragment from mitochondrial DNA based on the research of (Ayé et al., 2010). For those samples that were not successfully reproduced, primers L14995 and H16065 were used as complementary primers based on the research of Illera et al., (2020). The temperature protocol of PCR is given in Table S4. For qualitative analysis of PCR products, 1% agarose gel was used. The agarose gel was stained with one microliter of Green Viewer (Parstous brand) paint to be visible under UV light. Sequencing of PCR products was performed by the South Korean company Bioneer.

Nucleotide numbers
Sylvia cantillans species were used as outgroup (see also Olsson et al. 2013), In order to calculate a haplotype network, PopART 1.7 (Leigh & Bryant, 2015) software was used based on the TCS method (Keis et al., 2013).

Results
In total, we analysed 52 DNA sequences of Sylvia curruca from Qeshm Island. After aligning the sequences, 1057 nucleotide pairs from the cytb region were selected and combined with sequences publicly available in the Genebank. The tree of Bayesian inference (BI) shows six different clades, all with bootstrap values greater than 0.8 ( Figure 2). The clades are assigned to the taxa S. c. halimodendri, S. c. blythi, S. c. margelanica, S. c. curruca, S. c. althaea and S. c. minula. All samples from Qeshm belong to S. c. halimodendri. Within this subspecies, we found two clades based on differences in the DNA sequence (Table 1). The birds examined were scattered throughout the island and surrounding beaches during the winter season. All specimens examined were recorded in the same habitat.
In order to determine the genetic differences between the different subspecies of Sylvia curruca, the genetic distance was calculated and plotted based on the K2P replacement model as well as the haplotype network and approximately the same was obtained using TCS method (Table 2, Figure 3). The genetic distance between the subspecies is low (0.013-0.025), and it is only approximately 0.007 between the two clades found in Qeshm Island. The haplotype network, similar to the phylogenetic tree, 9 Zoology in the Middle East  confirm the existence of different clades of the Lesser Whitethroat subspecies in Qeshm Island. There highest number of evolutionary nodes was noted between the subspecies S. c. halimodendri and S. c. curruca.

Discussion
Our results show that Lesser Whitethroats wintering in Qeshm Island belong to the subspecies Sylvia curruca halimodendri, and is represented there in two clades. This indicates that birds from two different breeding populations of the same subspecies overwinter on the island. The subspecies S. c. halimodendri was first described from the border between deserts and steppes towards the northeast of the Aral Sea, from where it has apparently spread to parts of Kazakhstan and adjacent regions to the south (Shirihai et al., 2001;Olsson et al., 2013). The estimated breeding range of S. c. halimodendri is in Qinghai Province, China, and various localities in Kazakhstan (Olsson et al., 2013). Some birds from Qeshm Island belong to the same clade (clade 2) as No. KC512521 and KC512532 reported from Oman and China. They seem to breed from Kazakhstan to the northwest of the Xinjiang region (China) (see Table S1, S2). Therefore, the ten birds wintering on Qeshm Island probably come from the same breeding area. The upper clade (clade 1) of the phylogenetic tree, with 13 samples from Qeshm Island, may possibly be attributed to a population breeding in Mongolia and China. Votier et al. (2016) found a striking isotope difference between the populations inhabiting Siberian boreal forest (S. c. blythi) and the ones inhabiting semi-desert areas in Kazakhstan (S. c. halimodendri). This claim is similar to the findings of the present study because the phylogenetic tree and the genetic distance matrix confirm this difference between the two subspecies. Martens and Steele (1997) identified the songs of two birds from Kepe Dagh, Iran, as S. c. minula and Olsen et al. (2013) listed Iran as one of the main distribution areas of the subspecies S. c. minula. By contrast, Abdilzadeh et al. (2020) did not find any information about the occurrence of S. c. minula in Iran and our study also did not find any evidence for wintering of this subspecies in Qeshm Island. Abdilzadeh et al. (2020) showed a haplotype network with three taxa of the Lesser Whitethroat, in which S. c. halimodendri and S. c. althaea taxon were separated from the S. c. curruca taxon by the number of evolutionary nodes and an almost identical nucleotide polymorphism. Our results show that the two clades that include the Qeshm specimens (clade 1 and clade 2) were separated from the other subspecies of Sylvia curruca with a high evolutionary node and the TCS model confirms these results.

Supplementary Material
Supplementary Material is given as a Supplementary Annex, which is available via the "Supplementary" tab on the article's online page.