16S rRNA gene polymorphism supports cryptic speciation within the lesser blind mole rat Nannospalax leucodon superspecies (Rodentia: Spalacidae)

A Cryptic Subterranean Mammal Species, the Lesser Blind Mole Rat (Nannospalax leucodon syrmiensis)—Retreated but Not Extinct

“…They described the species Nannospalax syrmiensis (Méhely 1909) with the karyotypic formula 2 n = 54, NF = 90 as a group of seven populations endemic to Serbia, inhabiting the plains of the Srem district and the right bank of the Sava and Danube rivers from the confluence of the Drina, with the Sava in the west and the Great Morava with the Danube in the east [ 11 ]. Later, the nomenclature Nannospalax leucodon syrmiensis was accepted and widely used in the literature [ 6 , 8 , 10 , 14 , 15 , 16 , 21 , 26 ].…”

“…Total DNA was extracted from finger parts of living animals or from liver and muscle tissue using the DNeasy Blood and Tissue Kit, Qiagen. DNA isolation from teeth was carried out at the Institute of Forensic Medicine according to the procedures described in [ 14 ]. Universal primers [ 34 ] and seminested PCR were used for the amplification of ~600 bp long 16SrRNA gene fragments, as described in [ 14 ].…”

“…DNA isolation from teeth was carried out at the Institute of Forensic Medicine according to the procedures described in [ 14 ]. Universal primers [ 34 ] and seminested PCR were used for the amplification of ~600 bp long 16SrRNA gene fragments, as described in [ 14 ]. To obtain longer sequences of DNA extracted from old teeth, MT-CYTB gene parts were amplified with several primers that had been newly designed and modified according to [ 35 , 36 ].…”

“…They are known as typical inhabitants of the steppe-like grasslands, mountain steppes, and sand steppes, and they avoid marshy areas and quicksand [ 18 ]. Breeding experiments that revealed the complete reproductive isolation of seven N. leucodon CFs [ 8 , 11 , 19 ], substantiated by the results of molecular phylogenetic investigations [ 14 , 15 , 16 , 20 , 21 ], strongly suggest their cryptic-species status. The extent of their evolutionary divergence resembles that recorded among clearly distinguishable Spalax species [ 14 , 21 ].…”

“…MtDNA genes were selected instead of nuclear genes to increase the success rate of sequencing from old archived material. The mentioned methods have already proven to be excellent for the identification of BMR cryptic species [ 14 , 15 , 16 , 21 ]. The 16SrRNA gene is regularly used to identify well-differentiated species, genera and distantly related taxa [ 27 , 28 , 29 ].…”