Molecular evidence that Carex songorica Kar. & Kir. and C. gotoi Ohwi (Cyperaceae) are distinct species

Carex songorica and C. gotoi have similar appearance, but different distributions that almost do not overlap. Some authors considered C. gotoi as a subspecies of C. songorica. This opinion was based on the premise that C. gotoi differs from C. songorica only by slightly narrower utricles with thickened costate veins and somewhat longer beak. In order to clarify the extent of differences between these two taxa, we performed a molecular genetic study based on specimens from Asian Russia. We also re-examined herbarium specimens in order to clarify the correctness of identification and the differences between C. songorica and C. gotoi. Our results suggest that these taxa can be considered distinct species.

differs from C. songorica only by slightly narrower utricles with thickened costate veins and somewhat longer beak.
In order to clarify the extent of differences between these two taxa, we performed a molecular genetic study based on specimens from Asian Russia. We used three markers: a fragment of the plastid matk gene, and two fragments of the ribosomal RNA cluster, the external transcribed spacer (ETS) and the internal transcribed spacer 2 (ITS2). These sequences were successfully used in many studies on sedges, e.g., for the closely related section Vesicariae [6]. We also re-examined herbarium specimens in order to clarify the correctness of identification and the differences between C. songorica and C. gotoi.

Materials and methods
Herbarium specimens were taken from the NSK Herbarium. For C. songorica, we sampled 10 specimens from distant geographic locations and for C. gotoi, 4 specimens. Details on the specimens are given in Table 1. Dried leaves (100-500 mg) were ground in a mortar to fine powder and transferred to an Eppendorf tube with 1 ml of CTAB buffer (3% CTAB, 1.4 M NaCl, 30 mM Tris-HCl (pH 8.0), 2 mM EDTA) for 4 h at 65 °C. After incubation, 1 ml of chloroform was added and mixed, and the tube was centrifuged for 5 min at 16 000 g. The supernatant was transferred to a new tube and mixed with an equal volume of isopropanol, incubated for 5 min and centrifuged for 10 min at 16 000 g. The resulting pellet was dissolved in distilled water and purified on BioSilica columns (Russia) to remove residual polyphenols interfering with PCR reactions.
DNA amplifications were performed using commercial PCR mix (Biolabmix, Russia). A fragment of the plastid matk gene was amplified using universal primers matK-1 (5′-TTCAA [6]. All DNA fragments were sequenced from both ends. DNA chromatograms were edited using Chromas v.2.6.6 (Technelysium Pty Ltd). Phylogenetic trees were constructed for the concatenated dataset using MEGA X [7]. For the Maximum Parsimony (MP) algorithm, the Subtree-pruning-regraphing search with 1000 bootstrap repetitions was performed.

Phylogenetic Analyses
For all studied specimens, we obtained sequences of matk (591 bp), ITS2 (438 bp), and ETS (593 bp). No indels were found in any of the genes. The plastid matk sequences of C. songorica and C. gotoi were identical. Four polymorphic sites were found in ITS2, and nine, in ETS ( Table 2). Most of these positions delimited C. songorica from C. gotoi.
Almost no genetic diversity was detected within C. gotoi; only the C253 had one degenerate position in ETS. For C. songorica, on the contrary, many specimens had positions with the variants characteristic for C. gotoi.
Both ITS2 and ETS are parts of the ribosomal RNA cluster, and are represented as hundreds of tandemly repeated copies in the genome. Thus degenerate nucleotides represent not two alleles of a single locus located on different chromosomes, as we would normally expect, but the ratio of sequences with different variants within the cluster.
On the phylogenetic tree ( Fig. 1) one can see that both taxa form two reciprocally monophyletic groups. As seen in Table 2, C. songorica differs from C. gotoi by as much as four positions in ITS2 and eight, in ETS. This is higher than for many species of sedges. E.g., the species C. vesicaria and C. vesicata differ by one and four substitutions in these loci, respectively (our data, not shown).

Morphological comparison
C. songorica differs from C. gotoi by having orange utricles with fine veins and shorter beak 0.5-0.7 mm long. C. gotoi is characterized by dark purple utricles with thicker costate veins and longer beak (0.8-1.2 mm).
Specimen C252, reported as C. songorica from Irkutsk oblast (Bayanday region, v. Khuty) in the "Flora of Siberia" [5] and in the "Check-list of the Vascular Flora of the Irkutsk region" [8]. Our molecular data indicates that it belongs to C. gotoi. Its morphology is also closer to the latter species.
The Siberian Flora [5] reports C. gotoi from Khakasia (Bidzha river). We consider this to be the result of an erroneous identification, because our specimens from Khakasia (С279, С280, 282, С250) from adjacent regions belong to C. songorica.
We can thus conclude that C. gotoi и C. songorica are characterized by morphological and genetic differences and have different distributions. Although many botanists [2,4,5] referred to C. gotoi as a subspecies of C. songorica, our results suggest that these taxa can be considered distinct species.