Cytotaxonomy of the Prosimulium (Diptera: Simuliidae) of Western Asia Author Adler, Peter H. Author S, Ümit Author Irin text Zoological Journal of the Linnean Society 2014 2014-05-20 171 4 753 768 http://dx.doi.org/10.1111/zoj.12150 journal article 10.1111/zoj.12150 0024-4082 5310762 PROSIMULIUM RACHILIENSE COMPLEX Prosimulium rachiliense is divisible into two cytoforms. Cytoform ‘A’ is characterized by fixation of IS-20 and IS-21 ; sex chromosomes based on the CIt region; and a suite of unique, albeit low-frequency, autosomal inversions. Cytoform ‘B’ is characterized by the standard banding sequence ( CIt not withstanding), sex determination based minimally on CIII, and its own complement of unique autosomal polymorphisms, although sharing IS-20 with cytoform ‘A’ (and P. frontatum ). Given the distance ( c . 1200 km ) between Armenian and Turkish samples of P. rachiliense , the chromosomal differences might be clinal. Yet, the differences – two fixed inversions, nonhomologous sex chromosomes (I vs. III), and only one shared rearrangement (IS-20) − suggest that ‘A’ and ‘B’ are distinct species. Intervening collections eventually could provide a test of this hypothesis. Our Armenian sites are as little as 120 km from the type locality of P. rachiliense ( Azerbaijan , Kalbajar ) and all of these locations are in the Caucasus Mixed Forests Ecoregion ( sensu World Wildlife Fund, 2013 ). We, therefore, regard Armenian popula- tions as conspecific with the type specimen, and we tentatively suggest that Turkish populations represent a separate species. Differentiation within cytoform ‘B’ appears at some sites. The frequency of IS-20 homozygotes changes over time at Site 8 (Sökmen), from predominantly inverted in early April to predominantly standard in late April. The seasonal shift in standard and inverted homozygotes suggests the presence of two entities – possibly different cohorts or homosequential sibling species ( sensu Bedo, 1979 ) with identical sex chromosomes; resolution of the problem will require additional and more frequent sampling (cf. Newman, 1983 ). Some populations in south-western Anatolia express a second Y chromosome that builds on the typical Y-linked CIII rearrangement by linking IIIS-2 to it. All southwestern Anatolian samples are fixed for IS-20, further suggesting differentiation from more northern Turkish populations. Lacking additional evidence, however, we provisionally view these south-western Anatolian populations as an example of clinal variation (IS-20) and sex-chromosome polymorphism. Other cytological rearrangements are infrequent in cytoform ‘B’, suggesting that chromosomal criteria are unlikely to determine if additional species are represented within cytoform ‘B’ until more material from additional sites is studied. Figure 11. Chromosome arm IIIL of Prosimulium rachiliense cytoform ‘A’ (male), showing the standard sequence. Limits of IIIL-21–IIIL-24 of P. rachiliense ‘A’, IIIL-25 of Prosimulium frontatum , and IIIL-26 and IIIL-27 of Prosimulium tomosvaryi are indicated by brackets. Fixed rearrangements are italicized and polymorphic rearrangements are in standard type. Abbreviations: C, centromere; hb, location of heteroband 84hb of P. tomosvaryi ; hc, location of extra heterochromatin (85hc) of P. tomosvaryi ; hc(r) and hc(t), extra heterochromatin of P. rachiliense ‘A’ and P. tomosvaryi , respectively; s, shield; t, triad. Previous workers (e.g. Terteryan, 1968 ) morphologically recognized both P. rachiliense and Prosimulium pronevitschae Rubtsov in Armenia , but Crosskey & Zwick (2007) found no convincing structural differences between them, providing justification for synonymy of the latter with the former. Nor did we find any chromosomal evidence for two species in Armenia ; populations of cytoform ‘A’ are chromosomally cohesive. The geographical proximity of cytoform ‘A’ to the type locality of P. pronevitschae (North Osetia, village Gizel′), which is about 260 km directly north of our sites and also in the Caucasus Mixed Forests Ecoregion, further supports the synonymy. Nonetheless, the possibility that true P. pronevitschae is a distinct species cannot be discounted without samples from the vicinity of its type locality. If P. pronevitschae is a valid species, the more remote possibility that it is conspecific with our Turkish populations some 1250 km to the south-west, on the edge of the Anatolian Conifer and Deciduous Mixed Forests Ecoregion ( sensu World Wildlife Fund, 2013 ), would require testing. Further compli- cating the taxonomy of P. rachiliense is the enigmatic Prosimulium gigas Rubtsov , described from bloodfeeding females taken at about 2000 m in the Russian Caucasus approximately 100 km north-west of the type locality of P. pronevitschae . Known only from the original females, the species is similar to P. pronevitschae , although larger with paler legs and a slightly broader frons ( Rubtsov, 1956 ). The limited number of chromosomally analysed populations notwithstanding, the possibility should be considered that previous records of P. rufipes in Turkey refer to P. rachiliense cytoform ‘B’ and that P. rufipes does not occur in Turkey . Prosimulium fulvipes (Edwards) needs to be evaluated as the possible identity of cytoform ‘B’ and perhaps of all previous records of P. rachiliense and P. rufipes in Turkey . Described from Bulgaria (Mount Rila), about 600 km west of our Turkish populations, it carries the oldest available name that might apply to cytoform ‘B’, and, at least on morphological grounds, it is questionably distinct from P. rachiliense . Table 2. Frequency of inverted constituents for chromosomal rearrangements of Prosimulium frontatum in northern Armenia

Site	5†	6‡
Females: males: unknown*	6:3:2	9:3:4
IS-20	1.00	1.00
IS-23	1.00	1.00
IS-24	0.82	0.69
IL-12	1.00	1.00
IIS-12	0.77	0.91
IIIL-25	1.00	1.00
Mean no. heterozygous inversions/larva	0.45	0.56

Fixed rearrangements are italicized and polymorphic rearrangements are in standard type . *Gender could not be determined in larvae with heavy microsporidian infections. †Includes two gender-undetermined larvae with microsporidium similar to Weiseria laurenti . ‡Includes four females and two males with mermithid nematodes, two gender-undetermined larvae with microsporidium similar to Weiseria laurenti , and two gender-undetermined larvae with double infections of mermithids and the microsporidium. Table 3. Frequency of homologues with chromosomal rearrangements in Prosimulium rachiliense cytoform ‘A’ in Armenia

Site	1	2	3	4†	7
Females: males	15:8	12:3	27:15	1:0	0:2
IS-20	1.00	1.00	1.00	1.00	1.00
IS-21	1.00	1.00	1.00	1.00	1.00
IL-10	0.15	0.23	0.18‡
IL-11	0.01
CI region§	*	*	*	*
IIIS 2° N.O.¶	0.02
IIIL-21	0.04	0.03	0.02
IIIL-22	0.04	0.03	0.02
IIIL 88hc(r)	0.03
Mean no.	0.48	0.60	0.38	0.00	0.00
heterozygous
inversions/larva

hc, extra heterochromatin. Fixed rearrangements are italicized and polymorphic rearrangements are in standard type . †Sex chromosomes could not be determined for the single female. ‡In Hardy−Weinberg equilibrium (ss = 29, si = 11, ii = 2, where s, standard and i, inverted; χ 2 = 0.48, df = 1, P > 0.05). §* = X 0 Y 1 (lack of pairing in CI region) or X 0 Y 0 (complete pairing). ¶Secondary nucleolar organizer (N.O.) at the junction of sections 79/80. Prosimulium rufipes s.s. is a more northern species. Our samples from Germany and Switzerland conform chromosomally to material of P. rufipes described (as Prosimulium inflatum ‘aff. 2’) by Basrur (1959) from central France (Grenoble) and the French Alps. The lectotype of P. rufipes presumably was collected in Germany , although no locality was given by Meigen ( Zwick & Crosskey, 1981 ). We take the chromosomal characteristics of our German material, shared with Basrur’s (1959) French samples and our Swiss sample, as representative of the lectotype . Thus, P. rufipes is defined by seven fixed inversions, none of which is shared with the P. rachiliense complex. Sampling west of Turkey is needed to resolve the relation of P. rachiliense cytoform ‘B’ with populations currently considered as P. rufipes (and its putative synonym Prosimulium conistylum ) in areas such as Italy and the Rif Mountains of Morocco . Populations of so-called P. rufipes in Morocco’s Rif Mountains ( Adler & Belqat, 2001 ) are chromosomally more similar to those of the P. rachiliense complex, although on cladistic grounds, this relationship is unfounded because no unique rearrangements are shared; yet, neither are any unique rearrangements shared with P. rufipes s.s . Rather, individuals of Moroccan P. rufipes more closely resemble those individuals of P. rachiliense cytoform ‘B’ that carry the standard sequence in all arms. We, therefore, associate previous records of P. rufipes in Morocco with the P. rachiliense complex while not addressing its specific status.