White-tailed deer and deer keds harbour Megatrypanum trypanosomes uncovered by TthCATL-PCR
Overall, 15 WTDs including 9 males (5 juveniles and 4 adults) and 6 juvenile females captured were clinically healthy, showing normal values of heart frequency (44 to 128, mean of 76, beats/min), breathing frequency (18 to 48, mean of 34, breaths/min), and body temperature (35.4 °C to 40.2 ºC, mean of 37.4 °C). Inspection of WTDs (Fig. 2) for ectoparasites revealed an abundance of deer keds in ~73% (11/15) of them, mainly in the ventral, inner legs, and inguinal regions of the WTDs. No skin or fur damage was observed in ked-infested WTDs. Twenty-nine keds preserved in ethanol were identified morphologically as Lipoptena mazamae (Fig. 2)  and confirmed by COI DNA barcoding (GenBank: MN756795).
Microscopy of Giemsa-stained blood smears and the microhematocrit technique were unable to detect trypanosomes in blood of the 15 WTDs examined. However, haemoculturing (Fig. 2) yielded a trypanosome infection rate of 20% (three positive cultures), and one culture (TCC2268) was established and cryopreserved. These findings are consistent with very low parasitaemia but positive haemocultures as previously reported for other Megatrypanum trypanosomes [23,25]. The Megatrypanum-specific assay, TthCATL-PCR, was employed aiming at the detection of trypanosomes in blood samples from WTD and in gut contents from the deer keds. PCRs were positive for 7 out of 15 WTDs (~47%), and 11 out of 29 deer keds (~38%), including keds from the three WTD positive by haemoculturing.
Characterisation of trypanosomes from blood and keds of white-tailed deer using CATL sequences
CATL DNA sequences (274 bp) obtained by TthCATL-PCR from blood samples and culture (isolate TCC2268) of WTD and deer keds share highest similarity with sequences of Megatrypanum trypanosomes. Sequences herein determined were aligned with those of other Megatrypanum trypanosomes (GenBank) and used to infer their genetic relatedness. Trypanosome sequences obtained from deer keds were virtually identical to those obtained from WTD blood (TCC2268), indicating that they belong to a single trypanosome species.
Network inferences of 100 CATL sequences of 274 bp, amplified by TthCATL-PCR (Fig. 3) or 53 larger CATL sequences of 477 bp (data not shown) generated highly similar networks. A divergence of ~2.0% in the small fragment of CATL sequences separated TCC2268 from T. sp. D30. Even though originating from different continents, these two deer isolates were much more related to each other than to trypanosomes from bovids (cattle, buffalo, sheep and antelopes), even those from Venezuelan cattle and buffalo . TCC2268 and T. sp. D30 were assigned to close but different genotypes, TthII H (which includes CATL sequences from the deer keds) and TthII C, respectively. TCC2268 diverged by 5.3% from T. melophagium (TthII D) and by 3.3% from T. theileri of cattle nested into TthII (Fig. 3B).
Although useful for assessment of genetic diversity [42,50-52], the small polymorphism detected in short CATL sequences generated by TthCATL-PCR may be insufficient to discriminate reliable lineages and genotypes, therefore, the description of novel genotypes must be supported by additional markers including conserved (SSU rRNA and gGAPDH), and polymorphic (ITS rDNA and SL rRNA) sequences [15,25,42,43,50,53].
Barcoding of deer trypanosomes through SSU rRNA sequences revealed a new Megatrypanum trypanosome in white-tailed deer
Our comparative analysis of V7V8 SSU rRNA barcodes comprised Megatrypanum trypanosomes from cervids (Venezuela, Germany, Poland, Croatia, Japan, USA), and a large data set of trypanosome sequences from bovids including: cattle (Brazil, Venezuela, Argentina, Colombia, Germany, Poland, Croatia, UK, Japan, USA), water buffaloes (Venezuela, Colombia, Brazil), antelopes (Cameroon, Tanzania), and bison (Poland). In addition, our analyses included sequences of Megatrypanum trypanosomes from the guts of tabanids (Brazil, Africa, Russia), hippoboscids (Croatia, Scotland), tsetse flies (Africa) and sand flies (Italy) (Fig. 4; Supplementary Table S2). SSU rRNA barcodes corroborated the distribution of cervid and bovid trypanosomes in both TthI and TthII lineages, whereas the branching patterns intra-lineages could not be resolved using exclusively these highly conserved sequences (Fig. 4). In addition, two sequences of an elk (elk 317, GenBank JX178200-201) from the USA diverged by relevant and equidistant genetic distances from TthI (~3.0% divergence) and TthII (~3.1%), apparently representing a new lineage of Megatrypanum (Fig. 4).
SSU rRNA sequences of the Venezuelan isolate WTD TCC2268 was highly similar (99.7%) to that of the isolate WTD A3 from the USA, both sharing 99.5% similarity with T. sp. D30 of fallow deer from Germany. Besides these three deer trypanosomes, TthII comprised Trypanosoma cf. cervi from North American elk (elk328), and isolates of red deer (Cel34), fallow deer (DdP18) and sika deer (Cn1) from Poland. In addition, TthII included trypanosomes from African antelopes and T. melophagium of sheep, all clustering close to the trypanosomes of deer, whereas trypanosomes of cattle from North and South America, Europe and Asia formed a more separated cluster (Fig. 4). Trypanosomes from these European deer nested into TthII diverged from TCC2268 by 0.3%–0.6% in highly conserved SSU rRNA sequences. For comparison, TCC2268 diverged by just 0.6% from the reference T. theileri TREU124 of cattle, thus reinforcing that these sequences are too conserved to clearly resolve the relationships of the recently diversified Megatrypanum trypanosomes. Regarding possible vectors, trypanosome sequences obtained from the guts of Brazilian, Poland and Russian tabanids and Central African Republic tsetse flies were all assigned to both TthI and TthII lineages [24,29,36].
The lineage TthI also harboured trypanosomes (misclassified) referred to as Trypanosoma cf. cervi identified in WTDs (WTD A1, A5, A21, A148, NL15) and elks (elk142, elk328, elk416, elk421) from the USA , and isolates referred to as T. cervi from Poland red deer (Cel14St), fallow deer (DdP287) and tabanids (Fig. 4). The trypanosome (TSD1) from the Japanese sika deer  is the deer trypanosome more genetically distant (~1.4% sequence divergence) from the isolate TCC2268 (Fig. 4). In addition, this lineage also comprises T. theileri of cattle from South America, Japan and the USA, and T. theileri-like trypanosomes of South American water buffalo and Polish wisent [24,25,29].
Phylogeny based on concatenated SSU rRNA and gGAPDH genes support the independent species status for the new trypanosome from Venezuelan white-tailed deer in the subgenus Megatrypanum
It was previously demonstrated that gGAPDH sequences are generally more variable than SSU rRNA sequences, thus being more suitable for a better differentiation between closely related trypanosomes such as those of the subgenus Megatrypanum [15,24,25]. Corroborating results using SSU rRNA sequences, gGAPDH sequence from the isolate WTD TCC2268 was closest to T. sp. D30 (~2.2% sequence divergence), and more similar to T. melophagium and trypanosomes of antelopes, all clustering together. Cattle isolates formed other group within TthII, separated from WTD TCC2268 by an average of ~ 4.4% sequence divergence.
Here, gGAPDH sequences of TCC2268 were compared with available sequences from species/genotypes of Megatrypanum and concatenated with SSU rRNA sequences. The inferred phylogeny (Fig. 5) displayed a highly congruent topology compared with those of independent SSU rRNA (Fig. 4) or gGAPDH sequences (data not shown). Together, phylogenetic positioning and genetic distances of WTD TCC2268 from other phylogenetically validated species of Megatrypanum allowed for the description of this new isolate as a novel species herein designated Trypanosoma (Megatrypanum) perronei sp. n. Unfortunately, most trypanosomes from cervids have been known merely by partial SSU rRNA sequences such as those reported from elk and WTD from the USA, and deer isolates from Japan and Poland included in our previous SSU rRNA analysis (Fig. 4). Results obtained in the present study are congruent with previous studies using SSU rRNA, gGAPDH, CATL and ITS rDNA sequences [15,24,25,42,50,53].
Small polymorphism between ITS1 rDNA sequences of T. perronei sp. n. of white-tailed deer from Venezuela and the USA
To better understand the relatedness of T. perronei sp. n. with its closely related trypanosomes of deer from the USA and Germany, we compared ITS1 rDNA sequences, which are much more polymorphic than SSU rRNA and gGAPDH sequences. A comprehensive analysis of 122 ITS1 rDNA sequences from Megatrypanum trypanosomes (Fig. 6) was carried out including sequences of trypanosomes from a range of deer species and geographic origin: WTD TCC2268 from Venezuela (n = 2 sequences); WTD A3 (n = 2), WTD A1 (n = 2), WTD A21 (n = 3), WTD NL15 (n = 3), elk 416 (n = 1), elk 142 (n = 2), elk 421 (n = 1), and elk 328 (n = 1) from the USA; TSD1 (n = 1) from Japanese sika deer; TC2 (n = 1) from Croatian red deer; and T. sp. D30 from German fallow deer (n = 2). These sequences were aligned with those from bovid trypanosomes: T. theileri of cattle (29 sequences of TthI and 37 of TthII lineage); T. theileri-like of water buffalo (n = 13); T. melophagium of sheep ked; and trypanosomes of the antelopes (n = 14) sitatunga, duiker and puku.
Confirming previous studies, ITS1 rDNA sequences of Megatrypanum trypanosomes were distributed in the TthI and TthII lineages (Fig. 6) regardless of originating from bovids or cervids; thus, corroborating data from this (Figs 3-5) and previous studies [12,15,24,25,53]. Our analysis supported 6 genotypes of TthI (IA-IF) and 9 of TthII (IIA-II I) (Fig. 6). ITS1 rDNA sequences of T. perronei from Venezuela and the USA shared highly similar sequences (~0.6% divergence), and were tightly clustered together supporting the genotype TthII H. Trypanosomes from European cervids assigned to the lineage TthII C were the German T. sp. D30 of fallow deer [12,25] that clustered with the highly similar Croatian trypanosome of red deer , and they diverged by ~17% from T. perronei. Regarding the relationship between cervid and bovid trypanosomes, ITS1 rDNA sequences of T. melophagium of sheep diverged only by ~18% from T. perronei while divergences above 24% separated T. perronei from trypanosomes of cattle (TthII A and TthII B) and African antelopes (TthII F, G, E, I) .
Interestingly, ITS1 rDNA sequence of a trypanosome obtained from the gut of an Italian sand fly  was virtually identical to those of T. sp. D30 from Germany . As expected, deer trypanosomes of TthI diverged from T. perronei by remarkable divergences in ITS rDNA (Fig. 6): ~46% from both TthI D (WTD and elk) and TthI E (elk), which are genotypes identified in deer sympatric with the WTD from which T. perronei sp. n. (isolate WTD A3) was obtained in the USA . As we showed with other molecular markers, the greatest distances of ITS rDNA (~49%) among deer trypanosomes was between T. perronei and the trypanosome from Japanese sika deer (TthI F).
Host-parasite-vector relationships and evolutionary history of cervid trypanosomes
Although host specificity of Megatrypanum trypanosomes remains to be clearly demonstrated, our findings provide additional support for relevant host–parasite-vector association in the evolution of these trypanosomes. Species diversification was likely shaped by evolutionary constraints exerted by ruminant hosts. In addition, vectors may be also involved in trypanosome host-restriction because deer flies (tabanids) and deer keds (hippoboscids) are highly associated to their cervid hosts. In agreement with this hypothetical evolutionary scenario, we demonstrated that deer keds taken from WTD exclusively harboured T. perronei sp. n., corroborating a previous suggestion that these flies can transmit, cyclically and/or mechanically, trypanosomes to cervids . L. mazamae occurs from south-eastern USA to South America [39,55], and is tightly linked to WTD, although this ked can eventually jump to phylogenetically close deer species . Deer flies, which have been experimentally proven to transmit deer trypanosomes, and deer keds have been implicated as vectors of cervid trypanosomes [21,29,31,36,38,56,57]. Recent studies report on DNA from deer trypanosomes in guts of sand flies and culicids [54,58], but their roles as vectors remains to be investigated.
Trypanosome cross-infections of cervids and bovids have not been confirmed experimentally or by molecular epidemiology [8,12,15,21,24,25,33,53,59]. In Venezuela, we found WTD infected with T. perronei sp. n. while sympatric cattle and water buffalo were found infected with T. theileri and T. theileri-like, respectively . Japanese sika deer were found infected exclusively with the T. sp. TSD1, whereas sympatric cattle were infected with T. theileri of both TthI and TthII lineages . Deer, cattle and sheep harboured host-specific trypanosomes in Croatia . All these findings, coupled with data herein reported, provide strong evidence that Megatrypanum trypanosomes exhibit a narrow host range or even host specificity. Each trypanosome species/genotypes were found in a single host species or in closely phylogenetically related hosts, those found in cervids were never detected in bovids although one host species can harbour trypanosomes of more than one species or genotype [12,23-25,53]. To date, reports of elks and WTD sharing trypanosome genotype  relied merely on DNA detection, and genuine infections remain to be demonstrated. Reports of T. cervi, originally in an elk , and subsequently in a range of deer including WTD, wapitis , mule deer , moose  and reindeer in the USA , and in European fallow, roe and red deer  must be molecularly confirmed.
It has been demonstrated by isoenzyme and karyotype analyses that the trypanosome found in Swedish reindeer differ from those found in moose, and both differed from cattle isolates, despite all these animals living in sympatry . Similarly, data from zymodemes suggested the existence of different species of Megatrypanum infecting distinct species of deer and cattle in Germany . The isolates of T. perronei sp. n. from Venezuela and the USA are closely related, but not identical. Interestingly, T. perronei sp. n. is more related to deer trypanosomes from Germany, Croatia, Poland and Russia, all nested into TthII lineage [15,29,31,54], than to trypanosomes found in sympatric WTD and elks (USA) nested into TthI, a lineage also harbouring a trypanosome of Japanese sika deer [12,16].
Our findings agreed with multiple and relatively recent crossings of the Bering Strait by cervids infected with Megatrypanum trypanosomes reaching North America from Eurasia, and from these regions dispersing through the world. Altogether, deer-trypanosome-vector associations and phylogeography support a plausible evolutionary scenario where WTD infected with the ancestor of T. perronei sp. n., likely infested by its tightly linked ectoparasite L. mazamae, were introduced from North America into South America through the Panama Isthmus, reaching this continent at the Pliocene–Pleistocene boundary [1,60]. Cervidae originated in Asia between 7.7 and 9.6 mya, and according to fossil records, deer did not cross the Bering Land Bridge to North America before 4.2 to 5.7 mya . South American cervids are thought to have originated from at least two invasion events by North American deer: firstly, by the common ancestor of all deer species endemic of South America during the Great American Interchange at the Early Pliocene (~3 mya); and more recently (~1.5 mya) only by WTD at the Pliocene–Pleistocene boundary [1,60]. Concordant with our data on Megatrypanum trypanosomes, host–helminth assemblages were also associated with an early dispersion of cervids and bovids from Eurasia into North America and then into the Neotropics . Also supporting the recent dispersion of cervids and their parasites, Plasmodium sp. from the South American pampas deer (Ozotoceros bezoarticus) is closely related to Plasmodium odocoilei of North American WTD, and these two species are estimated to have diverged just by 0.3–0.9 mya .
Growth behaviour and developmental forms of T. perronei sp. n. in early haemocultures and co-cultivated with insect and mammalian cells examined by light and scanning electron microscopy
In early (7-10 days) haemocultures of WTD blood, live flagellates (phase microscopy) exhibited a few trypomastigotes with large body length, pointed posterior ends and noticeable undulating membrane, alongside large transition forms between trypo- and epimastigote forms, and dividing epimastigotes (Fig. 2A). Flagellates of T. perronei sp. n. from early haemocultures seeded on monolayers of insect cell (Hi-5), at 25 °C, initially formed clumps of small and rounded forms attached to insect cell membranes; these forms increased in length to became epimastigotes that remained adhered by their flagella forming rosettes until released into the supernatant of cultures (Fig. 2B). The developmental forms of T. perronei sp. n. co-cultivated with Hi-5 insect cells very much resembled those reported for T. (Megatrypanum) spp. in the guts of the ked L. cervi taken from red deer  and T. melophagium adhered to the cells of gut walls of the sheep keds [15,21].
Epimastigotes of T. perronei sp. n. in log phase Hi-5 cultures (5 days) multiply intensively attached by their flagella forming large rosettes (Fig. 7a), which initially remained adhered to the insect cells and afterwards are released in the supernatant, where free epimastigotes became progressively abundant (Fig. 7a,b). Giemsa-stained epimastigotes showed the rounded kinetoplast adjacent and lateral to the central nucleus with an almost imperceptible undulant membrane, and a long free flagellum (Fig. 7a,b). In mid-log cultures (7 days), most epimastigotes became longer and thinner with a pointed posterior extremity (Fig. 7b). Stationary phase cultures (10 days) of T. perronei sp. n. exhibited variable forms, all with a long free flagellum, including some wider epimastigotes exhibiting more preeminent undulant membranes (Fig. 7c). Some forms became progressively shortened in their posterior ends giving origin to blunted forms (indicated by arrow heads) during the differentiation of epi- to trypomastigotes (Fig. 7 b,c) and, finally, to ‘rounded’ forms with a long flagellum (Fig. 7b,c), which most likely represented metacyclic trypomastigote forms (Fig. 7d). In contrast with the slow movement of long epimastigotes, these “rounded” forms are highly mobile, and resemble metacyclic trypomastigotes of T. theileri described previously in the guts of tabanid flies and stationary cultures . Overall, initial co-cultivation of T. perronei sp. n. with insect cells, at 25 ºC, showed flagellates resembling those of Trypanosoma (Megatrypanum) spp. present in the guts of L. cervi, the Old-World deer ked taken from red deer .
Scanning electron microscopy (SEM) of the mid-log cultures (7 days) of T. perronei sp. n. in Hi-5 cultures showed flagellates of variable length and shape (Fig. 7e-j): slender epimastigotes without a noticeable undulant membrane (Fig. 7e) become broader epimastigotes exhibiting a conspicuous undulant membrane easily detectable by SEM (Fig. 7f,i,j). Following the differentiation from epi- to metacyclic trypomastigotes, a range of transition forms (indicated by arrows heads) were observed, including flagellates with a pointed posterior end and swollen central region (Fig. 7f-h), which progressively turn into forms with a blunted posterior extremity until whole differentiation into bell-shaped flagellates with long free flagella (Fig. 7f,g,k), which correspond to the apparently ‘rounded’ metacyclic trypomastigotes observed by light microscopy (Fig. 7c,d).
Log phase epimastigotes from Hi5-cultures were seeded into monolayers of mammalian LLC-MK2 cells, incubated at 37 °C with 5% CO2, and after one to 5 days, cultures were examined by light microscopy of Giemsa-stained flagellates (Fig. 8a-f) and SEM (Fig. 8 g-j). In the supernatant of these cultures, slender epimastigotes gradually became wider (Fig. 8a,g - one day culture) and gave origin to large and wide transition forms (indicated by arrow heads) between epi- and trypomastigotes initially exhibiting wide bodies (Fig. 8b,c,h), and then becoming long and slender showing well-developed undulant membranes and pointed posterior ends (Fig. 8 e,f,i,j). Both large epi- and trypomastigotes are multiplicative forms (Fig. 8b,d). Long and slender forms with sharpened posterior ends and prominent undulant membranes (Fig. 8 e,f,i,j) resemble those present in early haemocultures (Fig. 2) as well as blood trypomastigotes of T. theileri of cattle and T. theileri -like of water buffalo [6,21,23,53,63] and T. cervi and T. cervi-like [2,6,10,38,64]. Intracellular rounded flagellates resembling ‘amastigotes’  could be observed inside mammalian cells (data not shown), but unquestionable demonstration of their intracellular development and differentiation requires further studies.
Taken together, cultures of T. perronei sp. n. showed large epi- and trypomastigotes typical of Megatrypanum trypanosomes present in both early haemocultures and mammalian cell cultures (Figs 2, 8), similar to previously reported in deer blood [2,6,10,38,64]. In addition, clumps of small rounded forms and epimastigotes detected in early co-cultures of T. perronei sp. n. in insect cell (Fig. 7) were quite similar to in guts of deer keds infected with T. (Megatrypanum) spp. , allowing for inferences about the morphological differentiation through the life cycle of T. perronei sp. n. in vertebrate hosts and putative vectors according to the predicted life cycle (Fig. 2). Before T. perronei sp. n., culture of one deer Megatrypanum trypanosome was obtained only for T. cervi of elk from the USA . Morphological comparison of T. perronei sp. n. blood trypomastigotes and epimastigotes from vector guts with corresponding forms of previously reported deer trypanosomes did not revealed species-specific features, thus corroborating the high morphological resemblance of all Megatrypanum trypanosomes [2,6,10,21,38,64].
Ultrastructural characterization of Trypanosoma perronei sp. n.
Transmission electron microscopy (TEM) of cultured T. perronei sp. n. revealed mitochondrion, Golgi, glycosomes, acidocalcisomes, flagellum and overall ultrastructural organization typical of trypanosomes. A set of features can be considered common of Megatrypanum trypanosomes: An abundance of acidocalcisomes (Fig. 9a,b) distributed throughout the cell body; a kinetoplast exhibiting long and weakly compacted DNA fibrils and, consequently, wide thickness (Fig. 9a-d), a noticeable spongiome comprising a network of tubules and contractile vacuoles near the flagellar pocket (Fig. 9c,e,f), and the absence of cytostome. This is the first time that a deer trypanosome is characterized by TEM, and the ultra-structural arrangement was similar to that showed previously for T. theileri [23,65].
Phylum Euglenozoa (Cavalier-Smith, 1981); Class Kinetoplastea (Honigberg, 1963); Order Trypanosomatida (Kent, 1880; Hollande, 1982); Family Trypanosomatidae (Doflein, 1951); Genus Trypanosoma (Gruby, 1843);
New species description
Trypanosoma perronei sp. n. Teixeira, Camargo and García
Type material: Hapantotype, culture of the isolate TCC2268. The isolate WTD A3, known just by partial ITS rDNA and SSU rRNA sequences obtained from the blood of WTD from USA, and deposited in GenBank as Trypanosoma sp. PJH-2013a isolate WTD A3, was herein designed as a genotype of T. perronei sp. n.
Vertebrate host: Odocoileus virginianus (Ruminantia, Cervidae).
Invertebrate hosts (putative vectors):Lipoptena mazamae (deer ked).
Habitats: the blood of WTD and the digestive tract of deer keds. Type locality: State of Anzoátegui, Venezuela (N10°07′08.95'', W64°38′23.80'), South America.
Morphology: Log-phase epimastigotes co-cultured with Hi-5 insect cells with bodies averaging 21.61±9.11 μm long and 2.35±1.03 μm wide (Fig. 7). Large and slender trypomastigotes of co-cultures with mammalian cells showing bodies averaging 11.13±2.86 μm long and 1.73±0.29 μm wide, conspicuous undulant membranes and long free flagella (Fig. 8).
Moleculardiagnosis: DNA sequences unique to T. perronei sp. n. deposited in GenBank under the following accession numbers: SSU rRNA (MN752212), V7V8SSU rRNA (MN752143), gGAPDH (MN756794), ITS1 rDNA (MN752208-MN752209), and CatL (MN747149-MN747155).
Species depository: Culture of T. perronei sp. n. is cryopreserved at the Trypanosomatid Culture Collection of the University of São Paulo, TCC-USP, which also includes Giemsa-stained smears of cultures in glass slides, and DNA samples from cultures, blood of infected WTD and gut contents of deer keds.
ZooBank registration: To comply with the regulations of the amended 2012 version of the International Code of Zoological Nomenclature (ICZN) , T. perronei sp. n. was registered in ZooBank, the online registration system for the ICZN, under the Life Science Identifier (LSID): urn:lsid:zoobank.org:act:22747D46-AD0F-4013-B2D0-2F51080CFD54.
Etymology: The name “perronei” was given as a tribute to Dr. Trina Mercedes Perrone Carmona, a Venezuelan biologist who contributed to the knowledge of animal trypanosomiasis and the progress of the veterinary sciences in Venezuela, and who died unexpectedly in 2008.
Remarks: Recent phylogenies have unveiled more than one trypanosome species infecting the Pan-American WTD (O. virginianus). Besides T. perronei sp. n. of the TthII lineage detected in WTD from Venezuela and USA, this species of deer was reported as hosts of a trypanosome of the TthI lineage, so far only detected by PCR in WTD and elks . Elks are hosts of the T. cervi and, apparently, can also harbour other trypanosomes, but so far were not found infected with T. perronei sp. n. The only trypanosome reported in a deer species endemic to South America is T. mazamarum, described in blood of the brocket deer in Argentina , and never cultivated or molecularly characterized.