Batrachoseps wakei, Sweet & Jockusch, 2021

Sweet, Samuel S. & Jockusch, Elizabeth L., 2021, A New Relict Species of Slender Salamander (Plethodontidae: Batrachoseps) with a Tiny Range from Point Arguello, California, Ichthyology & Herpetology 109 (3), pp. 836-850 : 839-846

Batrachoseps wakei , new species urn:lsid:zoobank.org:act:15A84C27-4042-4585-8D2A-843A5C02F0A8

Suggested common name: Arguello Slender Salamander Figures 2–7

Holotype.— MVZ: Herp:293138 (original number SSS 32478 ), adult male, collected under boards lying against the northwest wall of the abandoned U.S. Coast Guard housing block at Point Arguello on Vandenberg Space Force Base , Santa Barbara County, California, 34.588N, 120.648W, 32 m elevation, Samuel S. Sweet, 7 March 2010. Precise locality information for the holotype and other specimens is held by the collections in which they were deposited.

Paratypes.— 14 specimens. LACM : Herps:192005–192014 (original numbers SSS 33428–33437 ) , 1 male, 1 female, 8 smaller individuals of undetermined sex, MVZ : Herp:293140– 293142 (original numbers SSS 32706–32708 ) , 2 males, 1 female, Honda Point, S base of Destroyer Rock, 30 m elevation, 34.608N, 120.648W, 2.8 km N of type locality, Alice Abela , Morgan Ball , Christopher Evelyn , John LaBonte, and Samuel Sweet, 9 January 2011 ( MVZ series), 30 April 2012 ( LACM series); MVZ : Herp:293139 (original number SSS 32479 ; Fig. 2A ), adult male, collected under debris 40 m NNE of the holotype at 34.588N, 120.658W, 33 m elevation, Samuel S. Sweet, 7 March 2010 .

Referred specimens.— 17 specimens: CCBER: Herps :32844 (original number SSS 32223 ), abandoned Coast Guard housing structure 0.3 km E of Point Arguello, 35 m elevation, 34.588N, 120.648W, Wes Fritz, 8 May 2006 ; CCBER: Herps :32845 (original number SSS 32229 ), same locality as CCBER: Herps:32844, Nancy Sandburg, 22 May 2006 ; CCBER: Herps :32846 (original number SSS 32384 ), W side of railroad tracks, 1.4 km NE Point Arguello, 51 m elevation, 34.598N, 120.648W, Alice Abela, 1 March 2009 ; CCBER: Herps :32847–32857 (original numbers SSS 33415–33425 ), Honda Point , gravel pit at S base of Destroyer Rock , 30 m elevation, 34.608N, 120.648W, Alice Abela, Morgan Ball, Christopher Evelyn, John LaBonte, and Samuel Sweet, 26 March 2012 ; SBMNH:HE:2733 (original number SSS 32385 ), E side of railroad tracks, 1.3 km NE of Point Arguello, 53 m elevation, 34.598N, 120.648W, Christopher Evelyn , 1 March 2009 ; SBMNH:HE:2734–2735 (original numbers SSS 32476– 32477 ), W side railroad tracks, 0.5 km SE Arguello Coast Guard Station, 51 m elevation, 34.588N, 120.648W, Samuel Sweet , 1 March 2009 .

Diagnosis.— The species is assigned to the genus Batrachoseps in having elongate genioglossus muscles attached to the midsection of the mandible, a large dorsal fontanelle in the skull, four toes on the hind feet, and genetic attributes that assign it to that genus. It is assigned to the subgenus Batrachoseps and differs from members of the subgenus Plethopsis ( B. campi , B. robustus , and B. wrighti ) in having fused premaxillary bones and more trunk vertebrae (21–22 vs. 17–19). Batrachoseps wakei is a large, long-bodied member (to 67.6 mm SVL, with 21–22 trunk vertebrae) of the B. pacificus group ( Jockusch and Wake, 2002; Martínez-Solano et al., 2012), sharing with B. major and B. pacificus large body size, a dorsal pattern of longitudinally vermiculate tan markings on a medium brown ground color, few white iridophores either dorsally or ventrally, a tendency to show orange patches on the distal tail, and a pale venter ( Fig. 2 ). Batrachoseps wakei differs from both species in having a smaller head, with the eyes protruding laterally beyond the margin of the upper jaw ( Figs. 3–4 ), in having ca. 50% fewer melanophores on the throat and chest ( Table 2), in often having extensive patches of orange pigment on the distal half of the tail ( Fig. 2 ), in lacking melanophores in the peritoneal lining, and in genetic attributes described below. It further differs from B. pacificus in having a longer tail. It further differs from B. major in having proportionally longer limbs, larger feet, and a shorter tail ( Fig. 4 ). Batrachoseps wakei has a proportionally smaller head and longer tail than B. m. aridus, a morphologically distinctive lineage variably treated as a subspecies of B. major ( Wake and Jockusch, 2000) or as a species (e.g., AmphibiaWeb, 2020), the ventral trunk is pale, and the dorsal pattern is of pale longitudinal vermiculations rather than a dense suffusion of gold and silver iridophores as seen in B. (m.) aridus.

Other species in the B. pacificus group ( B. gavilanensis , B. incognitus , B. luciae , and B. minor ) occurring to the north in central coastal California are smaller, with proportionally smaller heads, shorter limbs, narrower feet, and longer tails. They are darker both dorsally and ventrally, with their venters typically being dark gray or black, often with conspicuous white guanophores. The final member of the B. pacificus group, Batrachoseps gabrieli , from the central and eastern Transverse Ranges, is a smaller, darker animal with more gracile proportions (narrower head, smaller limb interval resulting from longer limbs, and a long, thin tail), typically with lichenose coppery markings on the dorsum.

Batrachoseps wakei is nearly sympatric with the widespread congener Batrachoseps nigriventris , from which it differs in its larger size, proportionally larger head, limbs, and feet, proportionally shorter tail, and pale ventral trunk and tail ( Fig. 2B compares the two). The same features serve to distinguish B. wakei from Batrachoseps attenuatus and Batrachoseps gregarius .

There are several species in the subgenus Batrachoseps in the Tehachapi Mountains and Sierra Nevada that are unlikely to be easily confused with B. wakei . The largest and most robust species, B. stebbinsi , is the only other species to approach B. wakei in standard length. It differs in having 18–19 trunk vertebrae (vs. 21–22), longer limbs, and larger feet. Batrachoseps simatus overlaps with B. wakei in vertebral number (having 20–21 trunk vertebrae) and also often having a dorsal pattern of longitudinal tan vermiculations. However, B. simatus is a smaller, more slender species with proportionally smaller feet, a longer but thinner tail, and a medium gray venter (vs. light tan). Batrachoseps bramei is much smaller than B. wakei , with a proportionally longer head and fewer trunk vertebrae (18– 19). Batrachoseps relictus is also a much smaller species, with proportionally shorter limbs, smaller feet, and a longer tail than B. wakei .

There are also four smaller Sierran species, all belonging to the B. diabolicus group: B. altasierrae , B. diabolicus , B. kawia , and B. regius . These are all small (less than 50 mm SVL), relatively slender species with fewer trunk vertebrae (19–20, except 20–21 in female B. diabolicus ), shorter limbs, relatively small feet, and proportionally longer tails, and none is likely to be confused with B. wakei .

Description.— Batrachoseps wakei is a large, moderately robust species, the largest male in the series being 65.0 mm SVL and the largest female 67.6 mm SVL, with a rounded head that is not distinct from the neck; further, the eyes extend beyond the outline of the head when viewed from below ( Fig. 3 ). The head width to hind limb length ratio of eight males with SVL 50 mm ranges from 0.60–0.76, mean 0.66, and in the four females with SVL 50 mm it ranges from 0.66–0.77, mean 0.70. Hind limb length averaged 16.0% of SVL in males (range 14.0–18.0%) and 15.4% of SVL in females (range 12.9–15.0%). Relative tail length increases with body size, from 0.57 of SVL at 21 mm to 1.27 SVL in the largest male and 1.37 SVL in the largest female. Regenerating tails and limbs are relatively uncommon, being evident in only 2 and 1 of 32 specimens, respectively.

Five of seven males and three of four females have 22 trunk vertebrae; the others have 21. Costal groove counts (one less than vertebral numbers) are 24 individuals with 21 and 8 with 20 costal grooves. There are usually nine costal grooves between adpressed limbs. The pattern of skin grooves (indentations) on the head demarcating muscle masses is typical of the genus. Neither a mental gland nor a postiliac gland is evident. Six adult males have 5–8 (mean 6.3) enlarged premaxillary teeth that perforate the upper lip. Cleared and stained specimens (CCBER 32847–32849) have one sacral, two caudosacral, and 39–43 caudal vertebrae. These specimens have 11–14 premaxillary, 13–15 and 14–17 maxillary teeth, 5–7 and 6–7 vomerine teeth, and ca. 250 small teeth in the elongate parasphenoid patches ( Fig. 5 ). Small preorbital processes are present on the vomers.

Measurements of the holotype (in mm).— Standard length (¼ SVL, snout to posterior angle of vent) 64.3; tail length 80.0; maximum head width 6.8; snout to gular fold 10.8; head depth at posterior angle of jaw 4.9; eyelid length 3.0; eyelid width 2.1; anterior rim of orbit to snout 1.8; orbit diameter 2.0; separation of external nares 2.5; snout projection beyond mandible 0.4; snout to forelimb insertion 13.7; axilla to groin length 40.2; forelimb length 8.0; hind limb length 9.0; limb interval between adpressed limbs 22.0; width of right hand 1.6; width of right foot 3.0; length of third toe 1.4; body width behind forelimbs 5.3. There are 21 costal grooves between the limb insertions, and 11 between adpressed limbs. There are 12 premaxillary, 30 maxillary, and 13 vomerine teeth (left and right sides combined), plus about 250 small teeth in the paired parasphenoid patches. Eight premaxillary teeth are enlarged and pierce the upper lip.

Coloration (in alcohol).— Adult B. wakei in ethanol are uniformly medium brown above, slightly darker on the head and in the dorsal third of each costal groove. Melanophores are deployed in rings around the openings of the skin glands, with the degree of envelopment decreasing slightly on the upper sides and then abruptly at the ventrolateral line. Melanophores become punctate and many fewer on the venter, almost absent on the ventral midline of the throat and trunk, and slightly denser on the pectoral region, along the cloacal lips, and the venter of the tail. The limbs are pigmented like the body, dark dorsally and paler beneath. The iris is dark, and the eyelids have dense melanophores along their edges.

Coloration in life.— Life coloration was examined in 26 individuals, including three small juveniles (21.0, 28.4, and 29.0 mm standard length). The dorsal ground color was light brown, created by melanophores grouped tightly around the openings of each skin gland. These became less dense at the ventrolateral line, and sparse melanophores were tightly condensed on the ventral surfaces, which were light tan with a faint orange cast on the tail. There were tiny white guanophores on the snout, anterior half of the eyelids and lateral surface of the face, neck, and trunk that did not reach the middle third of the venter behind the pectoral region. The dorsal ground color was overlain on the neck, trunk, and tail by tan to dull golden iridophores distributed as longitudinal streaks, usually less concentrated on the dorsal midline and so appearing to form irregular pale dorsolateral bands. Overlying these were patches of coppery orange iridophores, increasing in extent posteriorly on the dorsum of the tail ( Fig. 2 ). These orange patches enlarge with size and may cover the entire dorsum of the distal third of the tail in large adults. There seems to be no sexual dimorphism in this feature. Most individuals had small orange patches dorsally on the thighs. The iris is black with two thin golden arcs bordering the dorsal third of the pupil.

Small juvenile B. wakei were uniformly dark brown dorsally and tan ventrally, without tan dorsal vermiculations or orange iridophore patches, but with dense, punctate white guanophores on all lateral surfaces.

Osteology.— CCBER 32847–32849 (55.8, 53.0, and 50.0 mm SVL) were cleared and stained for comparison with series of the most similar congeners, Batrachoseps major (Los Angeles basin and Orange County) and B. pacificus (Santa Cruz Island). The skull ( Fig. 5 ) is relatively wide, with a large fontanelle between the frontals and parietals, as is typical of the genus. The premaxillae are fused anteriorly, their frontal processes remaining separate and loosely meeting the median edges of the nasals and the anterior extensions of the frontals, which do not contact one another. There are prominent foramina in the anterior frontals for the nasolacrimal ducts. Small prefrontal bones are present in some specimens, embedded in connective tissue between the frontals and the frontal processes of the maxillae. The cartilaginous olfactory capsules are only slightly protuberant beyond the outline of the upper jaw. The maxillae and premaxillae describe an arc that is exactly one-half a circle extending slightly posterior to the middle of the eyes, with small teeth that are absent from the posterior 10% of the maxillae. The pterygoid cartilages are thin and rodlike, extending forward to underlie the rear margin of the eye. The short parietals are widely separated anteriorly but approach each other posteriorly. Crests are absent on the otic capsules, although shallow grooves accommodate the thin posterior processes of the slender squamosals. The quadrates are small, and the lower jaw elements are relatively thin and flexible. There appears to be no mineralized or bony rod on the columella. Ventrally, the vomers contact the inner surfaces of the upper jaw elements but are widely separate from each other. Posteromedially, the vomers are strongly developed and thickened, bearing 6–7 stout, posteriorly directed teeth. A small preorbital process is present on each vomer, extending laterally to approximately the midpoint of the internal nares; this is an unusual trait for the subgenus Batrachoseps ( Wake, 1966; Marlow et al., 1979). The body of the vomer also extends beyond the lateral margin of the internal nares. As a result, the internal nares are largely surrounded by bone, rather than being open posteriorly, which is the more typical condition in the genus. Paired subtriangular patches of ca. 120–130 tiny teeth lie on the ventral surface of the parasphenoid, becoming broader to the rear.

The hyobranchial apparatus (not illustrated) is typical for the genus, being entirely cartilaginous with a flattened basibranchial with thin, backward-sweeping, divergent radii anteriorly, and long epibranchials that angle up over the shoulder to terminate slightly behind the forelimb insertion. No urohyal is present.

There are 21–22 trunk vertebrae behind the atlas, only the last lacking ribs, and a single sacral followed by two caudosacral vertebrae. The tails have 39–43 caudal vertebrae, more of them ossified in larger individuals. The frequency of obviously regenerated tails (,10%) seems lower in B. wakei than in other species in the subgenus (e.g., B. major , ca. 33% [ Cunningham, 1960]; B. attenuatus , 25–74% [ Maiorana, 1977; Meneghin, 1977]).

The limb elements are relatively stout, but tibial spurs are generally absent, even on large adults. Phalangeal formulas are 1-2-3-2 for fore- and hind limbs alike, with the first digit barely free of the footpad in both ( Fig. 6 ). Terminal phalanges are not much expanded. Carpal and tarsal elements are typical for the genus save that in all three individuals examined, the ulnare and intermedium are separate elements bilaterally, as is the case in most B. major and B. pacificus examined.

Etymology.— The species is named in honor of David Burton Wake, Professor of the Graduate School and emeritus Director of the Museum of Vertebrate Zoology at the University of California, Berkeley, in recognition of his immense contributions to the knowledge of California salamanders, as well as his broader roles in both amphibian conservation and evolutionary studies in western North America. David Wake has set an example of scholarship and integrity that has inspired his colleagues and his many graduate students (of whom we are two) by his insightful and never routine work on the evolution of salamanders, spanning the range of morphology, systematics, ecology, molecular genetics, and phylogeography. The complexity of the genus Batrachoseps has been progressively revealed by his embrace of the most current analytical techniques, and this is one of a minority of the 22 species in the genus that he has not had a hand in discovering or naming. As Museum of Vertebrate Zoology Director from 1971–1998, David Wake nurtured the museum’s focus on discovering the diversity and evolutionary history of western North American tetrapods. He also played a critical early role in focusing attention on the emerging declining amphibian crisis (e.g., Blaustein and Wake, 1990) and, as a founder of AmphibiaWeb, in promoting a collaborative vision for the amphibian research community and making high quality information about amphibians easily accessible to all.

Habitat and distribution.— As far as is known, Batrachoseps wakei has a very restricted distribution on a narrow marine terrace centered on Point Arguello. It is known from the south rim of Honda Canyon to an old railroad siding north of Rocky Point, from the coastal cliffs inland to the Union Pacific Railroad tracks, at elevations of 20–60 m ( Fig. 7 ). This zone is no more than 400 m wide and 4 km long, comprising about 130 ha. Within this region, it has been found at four sites ( Table 1). The western end of the Transverse Ranges here juts out into the Pacific Ocean, extending up to 10 km farther west than does the rest of the local coastline from Point Sal south to Point Conception, and rises to 650 m at Tranquillon Peak 8 km inland. This results in strong but very local enhancement of onshore winds, fog, and rainfall. The prevailing NW wind transports beach sand up onto the terrace, and decomposition of the relatively thick low vegetation, dominated by non-native Iceplant ( Carpobrotus edulis ) and including stands of the arborescent Giant Coreopsis, Leptosyne gigantea ( Fig. 8 ), creates a dark, sandy loam soil that is not found elsewhere on the coastal terraces. This soil holds burrows, unlike the sand deposits on lessexposed terraces nearby. To the north the coast is mostly dune fields for 60 km, and south of Point Conception the coast turns eastward for over 100 km and experiences much less fog and rainfall.

The crest of the western terminus of the Santa Ynez Range at Point Arguello is composed of early Miocene rhyolitic tuffs of the Tranquillon Volcanics Formation, unconformably overlying the Rincon Shale and overlain by the Monterey Formation, both deepwater marine deposits ( Dibblee, 1950, 1988; Cole and Stanley, 1998). Volcanic activity corresponds to the transition from a converging (subducting) to a transform (sliding) boundary as the Farallon Plate contacted North America, culminating in the origin of the San Andreas Fault Zone ( Atwater, 1970), and dates to about 14 mya. The rocks composing Honda Point (also known as Point Pedernales and including Destroyer Rock, the site of the catastrophic grounding of seven U.S. Navy destroyers in 1923) are Tranquillon volcanics, while to the south Point Arguello is underlain by Monterey shale.

The amphibian and reptile community recorded at the site reflects the strong maritime influence. Batrachoseps nigriventris avoids the coastal terrace but occupies the canyon of Honda Creek to within 400 m of the nearest records of B. wakei , and also occurs in rocky soft chaparral within about 1.1 km to the east. Batrachoseps nigriventris has been found in sympatry with six species in the B. pacificus group ( B. gabrieli , B. pacificus , B. major , B. minor , B. incognitus , and B. gavilanensis ; Yanev, 1978; Wake, 1996; Jockusch et al., 2001; Jockusch and Wake, 2002), so sympatry with B. wakei where their habitats meet would not be surprising. The plethodontid salamanders Aneides lugubris and Ensatina eschscholtzii are each microsympatric with B. wakei , as is the frog Pseudacris hypochondriaca , with Rana draytonii in Honda Creek 0.5 km away. Among lizards, Sceloporus occidentalis , Plestiodon skiltonianus , Elgaria multicarinata , and Anniella pulchra are microsympatric with B. wakei . Among snakes, only Diadophis punctatus and Pituophis catenifer annectens have been recorded on-site, with Crotalus helleri nearby.

Genetic diversity in B. wakei . — Overall genetic diversity in B. wakei is low. Despite relatively large samples (18 mtDNA samples, 40–50 allele copies for each nuclear locus) spanning the full known range of the species, no variation within B. wakei was found in five of the seven analyzed markers, including the mitochondrial gene. The exceptions were rag1 and gapdh, each of which had three sampled alleles. Geographic structuring was present ( Fig. 9 ), with AMOVA on the multilocus data showing that 31.2% of the variation was distributed among populations (P, 0.001). When run on the individual loci, no structure was detected in gapdh (P ¼ 0.60), while 52.1% of the variation at rag1 occurred between populations (P, 0.0001).

Variation in gapdh involves the length of an intronic mononucleotide repeat, which has 8, 9, or 14 Gs; such motifs are expected to have a high mutation rate. Multiple alleles were observed in all populations ( Fig. 9 ). The 8-nucleotide repeat was private to one population in these samples, while the other two repeat lengths were observed in all four populations. Each of three rag1 alleles was found in at least two populations, and multiple alleles were sampled in three of four populations ( Fig. 9 ). The most widespread allele was fixed in the sample of seven specimens from the northernmost population.

Phylogenetic relationships of B. wakei . — Supplemental Table S2 (see Data Accessibility) gives alignment information and the selected models and partitioning scheme for each marker. Gene trees for each marker are shown in Supplemental Fig. S1 (maximum likelihood; see Data Accessibility) and Supplemental Fig. S2 (Bayesian; see Data Accessibility); haplotype networks are provided for loci where B. wakei formed part of a multispecies polytomy (Supplemental Fig. S3 ; see Data Accessibility). No allele sharing across species was observed, with the exception of one allele in pomc, the least variable locus. Additionally, species-level structuring was strong, with most species being monophyletic or nearly so at most markers. However, relationships strongly conflicting with monophyly of conspecifics were not uncommon.

In all species-tree analyses, B. wakei belonged to a clade comprising three species: B. major , B. wakei , and B. pacificus , which received high support in most analyses ( Fig. 1B ; Supplemental Table S3; see Data Accessibility). Relationships within this clade depended on inference method and were not strongly supported, with B. wakei inferred to be the sister taxon to a clade comprising the other two lineages in all analyses using ASTRAL and with it instead inferred to be the sister taxon to B. major with SVDQuartets (Supplemental Table S3; see Data Accessibility). This southern affinity of B. wakei is reflected in the relationships in five of seven gene trees or haplotype networks; in the other two loci (rag1, cytb), B. wakei shows central coastal, rather than southern, affinities (Supplemental Figs. S1–S 3 ; see Data Accessibility).

Figures

FIG. 2. Photos of Batrachoseps wakei. (A) Paratype (MVZ 293139, adult male, SVL ¼ 60.4 mm) in life; (B) paratype of B. wakei (MVZ 293140, adult male, SVL ¼ 63.0 mm) contrasted with B. nigriventris from Honda Canyon, Santa Barbara Co., California, in close parapatry with B. wakei; (C) ELJ 1662. Photos A, B by Sam Sweet; photo C Iñigo Martínez-Solano, used with permission.

FIG. 3. Head shape in ventral view of (A) Batrachoseps wakei (CCBER 32844, adult female, SVL ¼ 67.6 mm); (B) Batrachoseps major (SSS 34390, adult female, SVL ¼ 66.5 mm); and (C) Batrachoseps pacificus (SBMNH 654, adult female, SVL ¼ 67.0 mm); scale bar ¼ 5 mm.

FIG. 4. Comparisons of body proportions in B. wakei and its closest relatives. (A) Head length (snout to gular fold); (B) maximum head width; (C) hind limb length; and (D) tail length versus snout–vent length (SVL); color and shape indicate species.

FIG. 5. Skull of an adult female Batrachoseps wakei (CCBER 32848, SVL ¼ 53.0 mm); anterior ventral (top) and dorsal (bottom) views; bones, teeth, and cartilage (stippled) are shown. Scale bar ¼ 2 mm.

FIG. 6. Dorsal view of the right front and hind foot of an adult male Batrachoseps wakei (CCBER 32847, SVL ¼ 55.8 mm); cartilage is stippled. Scale bar ¼ 2 mm.

FIG. 7. Distribution of B. wakei. (A) Topographic relief map, with black dots marking the four localities at which B. wakei has been found; population numbers are as in Table 1. White dots indicate nearby localities for B. nigriventris on Vandenberg Space Force Base. Map tiles by Stamen Design, under a CC BY 3.0 license. Map data by OpenStreetMap under ODbL. (B) Inset of coastal region showing hypothesized range of B. wakei.

FIG. 8. Habitat of B. wakei. (A) Type locality at Point Arguello, California, USA; (B) northernmost locality at Honda Point, California, USA. Photographs Ivan Parr, used with permission.

FIG. 9. Haplotype networks and geographic variation in allele frequencies for the two loci showing variability in B. wakei. Haplotype networks for rag1 (A) and gapdh (B), with circle size scaled to allele frequency and shading indicating the four source populations. Distribution of alleles across geography for rag1 (C) and gapdh (D), with circle size scaled to sample size from the population, and shading distinguishing the three alleles.

FIG. 1. Distribution and relationships of the B. pacificus group. (A) Colored polygons show the ranges of the seven species currently recognized in B. pacificus (based on IUCN range polygons), and black dots show the locations of molecular samples included in this study; brown polygon shows range of B. nigriventris. Arrow and purple circle indicate the locality of the species described here. Map tiles by Stamen Design, under a CC BY 3.0 license. Map data by OpenStreetMap under ODbL. (B) Species tree for the B. pacificus group and selected outgroups inferred from coalescent analysis of seven nuclear and one mitochondrial marker. Support values are ASTRAL-III’s local posterior probabilities; branch lengths are in coalescent units. See Data Accessibility for tree file.