Taxonomic revision of the flea genus Agastopsylla Jordan & Rothschild 1923 (Siphonaptera: Ctenophthalmidae)

: Fleas of Argentina are receiving renewed systematic interest, but the identification of many species associated with small mammals can be problematic. We review the taxonomy of the flea genus Agastopsylla including the re-description of two species and one subspecies, and designate neotype and neallotype for Agastopsylla hirsutior , neotype for Agastopsylla nylota nylota from the “Colección Mamíferos Lillo Anexos” (CMLA), Universidad Nacional de Tucumán, Argentina, and neotype and neallotype for Agastopsylla pearsoni from the Natural History Museum (London, U.K.). Additionally, a key to identification of the species of Agastopsylla and a distribution map of the species of the genus are included.


INTRODUCTION
The genus Agastopsylla Jordan & Rothschild 1923 (family Ctenophthalmidae, subfamily Ctenophthalminae) is characterized by the reduction in size and the coloration of the spines of the genal comb (Hopkins & Rothschild 1966). The distribution of the genus is restricted to Perú, Chile and Argentina (Hopkins & Rothschild 1966) and the known species are associated mainly to cricetid rodents (Hopkins & Rothschild 1966, Lareschi et al. 2016 Beaucournu et al. 2011, Lareschi et al. 2016. In Argentina, only two species and two subspecies, A. b. boxi, A. b. gibbosa and A. pearsoni (Lareschi et al. 2016) have been recorded.
There are several issues that hinder the systematic study of the genus Agastopsylla. For example the loss of type specimens in reference collections, or the incomplete original descriptions of some species described more than 60 years ago, and the lack of voucher specimens deposited in systematic collections.
We here provide a review of the genus Agastopsylla, which includes the re-description of two species and one subspecies. In accordance with Article 75 of the International Code of Zoological Nomenclature (ICZN 1999), we designate neotype and neallotype for A. hirsutior and Agastopsylla pearsoni and neotype for A. n. nylota and include the first description of the male of A. hirsutior and of the female of A. n. nylota. Additionally, a key to identification and a distributional map of the species of Agastopsylla are also included.

MATERIALS AND METHODS
Fleas were obtained from rodents collected in the provinces of Salta and Tucumán, Argentina. The rodents were captured with Sherman live traps baited with peanut butter and oats. Fleas were removed with forceps and brushes, prepared following conventional techniques for taxonomic identification (Hastriter & Whiting 2003) and photographed with a digital camera AxioCam ERC5S and processed with Microscope Imaging software ZEISS ZEN 2012-Blue edition (Germany). For the identification of the taxa, the original descriptions of the species and the identification keys (Johnson 1957, Hopkins & Rothschild 1966, Beaucournu et al. 2011 were used. Anatomical terms were adapted from Rothschild & Traub (1971)  Neotypes and neallotypes of the species of Agastopsylla from Argentina were deposited in the "Colección Mamíferos Lillo Anexos" (CMLA), Universidad Nacional de Tucumán, Argentina. Mammal nomenclature follows Wilson & Reeder (2005), Barquez et al. (2006) and Patton et al. (2015). The host specimens were deposited in the Colección Mamíferos Lillo (CML), Universidad Nacional de Tucumán and in the Sam Noble Oklahoma Museum of Natural History (SNOMNH), University of Oklahoma (Norman, OK).
We used the morphological characters observed in this study as well as the published morphological information for species and subspecies (Jordan & Rothschild 1923, Traub 1952, Beaucournu & Alcover 1990, Sanchez & Lareschi 2014 to formulate supportive illustrations for the characters in the identification key. To illustrate the distribution of the genus Agastopsylla, the coordinates of the localities recorded for the species were obtained from the literature and the missing coordinates in the bibliography were determined using Google Earth Pro Version 2018. The map was designed with ArcGis 10.1 program (ESRI 2011 (Sanchez & Lareschi 2013, 2018.
Morphologically, A. boxi gibbosa is only known by the shape of sternite IX of the male, and females of the two subspecies are indistinguishable (Beaucournu & Alcover 1990).

Emended diagnosis
Agastopsylla hirsutior is unique by the following combination of characters: lateral surface of the hind tibia densely covered by 22-24 setae (Figs. 2a,b), the male telomere notably short; lateral margins converge towards the apex (Fig.  2c). Female spermatheca with a globular bulga which bears a small dorsal pump (Fig. 2d).

Redescription
Head. Front margin convex with two placoid pits and one row of four short setae. Behind this row and on the surface of the gena, several short setae are distributed; ventral margin of the gena with two long setae. Antennal scape with six setae, pedicel with one row of seven short setae on posterior margin and male clave slightly larger than that of female; border of fossa antennal with one row of 5-6 small setae. One seta anterior to eye. Eye reduced but pigmented. Genal comb pale with five spines, first spine most ventrally located, very small and poorly visible. Maxilla fusiform, labial palpus with five 76º2'9.15"W. Agastopsylla nylota euneomys ( ) (Lewis & Spotorno, 1984) CHILE: RM Region Metropolitana: Farellones, 2,800 m, 33º21'00.00"S, 70º20'00.00"W. Agastopsylla pearsoni (•) (Traub 1952). PERU: Puno Department, San Antonio de Putina Province: Picotani 4,537 m, 14º32'59.67 "S, 69º48'0.02"W.
segments extending to the base of the forecoxa. Occiput with three rows of setae, first and second rows with four short setae and third with six long setae plus intercalaries; additionally, there are two long setae near antennal fossa.
Thorax. Pronotum with two rows of setae: an anterior with six short setae and main row with five long setae plus intercalaries, and between these two rows a small seta. Pronotal comb with eight spines. Mesonotum with three rows of setae: first with three short setae, second with six short setae and main row with five long setae plus intercalaries. Metanotum with four rows of setae: first three rows with short setae and fourth with one row of seven setae plus intercalaries. Mesepisternum with an anterior group of small setae and two long posterior setae. Mesepimeron with five setae, two thin and three thicker; lateral metanotal area with one long seta; pleural arch and ridge well developed. Metepisternum with one long seta and four short setae. Metepimeron with nine setae scattered over the surface, seven thin and short and three thick and long.
Legs. Forecoxa with 40-45 setae scattered over surface and one seta on posterior margin; mesocoxa and hindcoxa with setae scattered only on the posterior margin. Forefemur with two small setae on the dorso-posterior side and one seta on the ventro-posterior margin.
Abdomen. Tergites without spinelets. Tergites I-V with three rows of setae: first and second rows with short setae and the third row with long setae. Tergites VI-VII with two rows of setae, anterior with short setae and the posterior with long setae. Tergite VII (T-VII) with one antesensilial bristle. Sensilium with 13 sensilial pits. Sternites III-VII with one main row of three setae in the male, and four setae in the female.
Modified abdominal segments, male ( Fig.  2c): Sternite VIII (S-VIII) with three long setae and five short setae on the posterior region. Basimere dorsally rounded with posterior margin slightly convex and dorsal margin with three long thick setae and four thin setae; telomere with anterior and posterior margins convergent towards the apex with posterior margin with several setae. The manubrium is narrow and noticeably sharp at apex. Proximal arm of sternite IX (S-IX) narrow in the base and widened at the apex. Distal arm of S-IX (DA9) uniformly narrow with apex strongly rounded, posterior margin convex with some setae, and a few setae over surface close to apex.
Aedeagus. Median dorsal lobe dorsally convex. Sclerotized inner tube very short. Crochet short and posteriorly sharp. Fulcrum well developed with elongated medial and latero-ventral lobes; latero-ventral lobe strongly sclerotized. Crescent sclerite slightly sclerotized, narrow and long, subequal in length than the fulcral latero-ventral lobe. The dorsal extension of median lamina of aedeagal apodeme strongly sclerotized. Penis rods extended beyond the apex of aedeagal apodeme and uncoiled Modified abdominal segments, female: Tergite VIII (T-VIII) with 13 setae scattered over surface. Length of anal stylet four times the length of its width (at base); with a long apical seta. Ventral portion of S-VII convex. Dorsal and ventral anal lobes triangular shape, both with one long seta at the apex. Hilla of spermatheca longer than bulga; bulga globular with a small dorsal pump; cribriform area noticeably sclerotized; duct of spermatheca expands into a bursa copulatrix, which bears a hyaline perula and a visible duct (Fig. 2d).
Remarks. Previous to our study, A. hirsutior was known only by the single female holotype deposited in the Field Museum of Natural History, Chicago. The holotype could not be found and is considered lost. The newly acquired male and female were collected from the highland pasture (2830 m) in the last stratum of the ecoregion Yungas Forests. The species was recorded for the first time on A. jelskii from Peru; in the present study a male and a female were documented from A. spegazzinii, constituting a new distribution and flea-host association.
We designate herein the male as the neotype and the female as the neallotype. Each is described above and an emended diagnosis is provided. The accompanying female associated with the neotype is conspecific to the description and illustrations of the Peruvian female holotype. The male provides a more distinctive morphological representation of the species than the female and is therefore designated as the neotype.  (Fig. 1).

Redescription
Head. Front convex with two placoid pits and micro-punctuations scattered over surface. In male, front with one row of five setae and behind this, on the surface of the gena, several short setae; in female front with several minute setae, but not row of setae. Antennal scape larger in the male and both sexes with six setae; pedicel with one row of 10 short setae on posterior margin and clava slightly larger in male; border of antennal fossa with one row of 12 small setae in male, and eight small setae in female. One seta anterior to eye. Eye reduced but pigmented. Genal comb pale with four or five spines in male and four in female, the first most ventral very small. Ventral margin of gena with two long setae. Maxilla fusiform; the labial palpus with five segments, extending to the base of the forecoxa. Occiput with micro-punctuations scattered over surface and three rows of setae, first and second rows with four short setae and third row with six long setae plus intercalaries; additionally two long setae near the antennal fossa.
Thorax. Pronotum with one main row of 5-6 setae plus intercalaries. Pronotal comb with 10 spines in male and eight in female. Mesonotum with three rows of setae: first row with three short setae, second with six short setae and main row with five long setae plus intercalaries. Metanotum with three rows of setae in male, the first two with short setae and the third with one row of six setae plus intercalaries. Female with two rows of setae, first row with eight setae and second with six setae plus intercalaries. Mesepisternum with an anterior group of small setae and two long posterior setae. Mesepimeron with seven setae, two short and five long. Lateral metanotal area with one long seta and two small setae next to well developed pleural arch; pleural ridge well developed. Metepisternum with long seta and five short setae in posterior region. Metepimeron with two rows of setae, anterior with four setae and posterior with 3-4 setae.
Abdomen. Tergites without spinelets. Tergites I-VII with three rows of setae; first row with 3-4 short setae, the second with 6-8 short setae and the third with seven long setae plus intercalaries. Tergite VII with one antesensilial bristle. Sensilium with 11-12 sensilial pits.
Sternites III-VII with one main row of three setae in male, and four in female.
Modified abdominal segments, male: Sternite VIII with 8-10 long setae next to the posterior region. Basimere narrows at apex with two long setae near apex, posterior margin convex with a long seta and three small thin setae below. Telomere noticeably longer than that of A. hirsutior and anterior and posterior margins are parallel (Fig. 3c) with several setae near apex and ventro-posterior margin. The manubrium is narrow and noticeably sharp at the apex. Distal arm of S-IX as in A. hirsutior but with a thicker sclerotization in the lower portion of the posterior margin.
Aedeagus. Median dorsal lobe dorsally convex with a sharp dorso-posterior projection and an undulating anterior margin. Sclerotized inner tube very short. Crochet longer than in A. hirsutior. Lateral lobe with a convex portion in the middle. Fulcrum well developed. Aedeagal apodeme sharp in the apex. Penis rods long and uncoiled.
Modified abdominal segments, female: Tergite VIII with two setae (one greatly reduced) below the spiracle and 18 setae scattered over surface, with setae next to ventral margin smaller than the remaining. Length of anal stylet is onethird the total length of its apical seta. Sternite VII convex in middle (Fig. 3d). Dorsal and ventral anal lobes triangular shape, both with one long seta at the apex. The spermatheca was missing.
Remarks. The male holotype was deposited in the Field Museum of Natural History, Chicago, and could not be located. The female of A. nylota nylota was unknown prior the single female collected in our study. Although the spermatheca is missing, its occurrence with three males from the same host (CML 8044), presence of the same number of setae on the dorsal margin of the hind tibia, similar chaetotaxy to the male, and the presence of four genal spines are evidence that the female and males are of the same taxon.
One of the specimens examined (male) was previously identified as Agastopsylla pearsoni by López-Berrizbeitia et al. (2013) but further studies and comparisons with specimens deposited in collections allowed us to reidentification of the specimens as A. n. nylota. When A. nylota nylota was recorded for the first time in Peru, the hosts were not identified and in the original description the authors suggested three possible hosts (see in type host and locality); it is necessary to emphasize that the distribution P. darwini is restricted to Chile (Steppan & Ramírez 2015). Thus, this second record of A. nylota nylota in the present study represents the first confirmed association with P. osilae. The subspecies was collected in the Monte Desert of Mountains and Isolated Valleys eco-region (2680 m).
Remarks. This subspecies is only known by the holotype male deposited in the U.S. National Museum, Washington, D.C. Agastopsylla nylota euneomys differs mainly of the nominate subspecies in the following characters: genal comb of 3 pale spines, labial palpus extending well beyond apex of forecoxa and telomere shorter and wider (Lewis & Spotorno 1984).
Remarks. The holotype male and allotype female, deposited in the Field Museum of Natural History (Chicago) could not be found and are considered lost. Therefore, we designate herein the paratype male as the neotype and the paratype female as the neallotype. These paratypes are deposited in the Natural History Museum (London, U.K.).

DISCUSSION
In the present revision of the genus Agastopsylla for Argentina, we add A. hirsutior and A. n. nylota, and remove A. pearsoni from the fauna of the country. Hopkins & Rothschild (1966), who realized a complete revision of the genus, at that time, were unable to review specimens of A. hirsutior and A. nylota nylota. Although two of our designated neotypes are not from the type localities of the original, their designation is important and necessary to maintain nomenclatural stability and to solve problems of doubtful and confusing identities in the future. The finding of A. hirsutior in Tucumán Province extends its geographic distribution ̴ 1700 km southward, and the record for A. n. nylota in Salta Province also extends its distribution ̴ 2700 km southward, from the records previously documented by Traub (1952) in Peru (Caccachara, Dep. Puno and Carhuamyo, Dep. Junin, respectively). These results are consistent with what was expressed by Lewis & Spotorno (1984), who assumed that the species of Agastopsylla range much wider than indicated by collection records considering mainly the large numbers of small cricetids rodents having Andean distributions.
In  Beaucournu et al. 2014, Sanchez & Lareschi 2014, confirm that it is mainly distributed in the Andean biogeographic region (as defined by Morrone 2015) (see Fig. 1), as do several species of the flea fauna of northwestern Argentina, among those Neotyphloceras crassispina, Nonnapsylla rothschildi, Plocopsylla hastriteri and Tiarapsylla argentina (Lareschi et al. 2011, López-Berrizbeitia et al. 2015, 2018. In order to morphologically distinguish the species the flea specialists, have mainly and traditionally used the number of setae in the outer side of the hind tibia, and characters of the genitalia; among these, the shape of telomere in the males and the shape of spermatheca in females (Traub 1952, Beaucournu & Alcover 1990, Beaucournu et al. 2011. Additionally the aedeagus, in the male, has been considered as one of the most important diagnostic characters (Lewis & Spotorno 1984, Sanchez & Lareschi 2014. In this study, we used mainly these characters to distinguish species and subspecies. More collecting and studies of these fleas are needed to define the morphological characters of the female of A. n. nylota and, complementary, comparisons with materials deposited in collections are required to identify the species with confidence. Specimens of genus Agastopsylla are hard to find and therefore are poorly represented in collections. Several authors have suggested that they are nest-fleas (Traub 1952, Johnson 1957, Hopkins & Rothschild 1966, so they have developed certain morphological adaptations as are the partial fusion of the metepimeron with the metanotum, reduction of the eyes, and the elongated labial palpus and mouthparts. These characters are all shared with other genera known to be nestfleas (Lewis & Spotorno 1984). It is important to increase the sampling efforts as well as to implement complementary collecting methods such as searching in nests and shelters, and depositing the voucher specimens in systematic collections, to ensure their proper study and preservation.