Discovery of the Largest Orbweaving Spider Species: The Evolution of Gigantism in Nephila

Background More than 41,000 spider species are known with about 400–500 added each year, but for some well-known groups, such as the giant golden orbweavers, Nephila, the last valid described species dates from the 19th century. Nephila are renowned for being the largest web-spinning spiders, making the largest orb webs, and are model organisms for the study of extreme sexual size dimorphism (SSD) and sexual biology. Here, we report on the discovery of a new, giant Nephila species from Africa and Madagascar, and review size evolution and SSD in Nephilidae. Methodology We formally describe N. komaci sp. nov., the largest web spinning species known, and place the species in phylogenetic context to reconstruct the evolution of mean size (via squared change parsimony). We then test female and male mean size correlation using phylogenetically independent contrasts, and simulate nephilid body size evolution using Monte Carlo statistics. Conclusions Nephila females increased in size almost monotonically to establish a mostly African clade of true giants. In contrast, Nephila male size is effectively decoupled and hovers around values roughly one fifth of female size. Although N. komaci females are the largest Nephila yet discovered, the males are also large and thus their SSD is not exceptional.


Introduction
The origin and maintenance of sexual size dimorphism (SSD) are much debated topics in evolutionary biology [1,2,3]. Spiders in general [4,5,6,7,8], and the orbweaving family Nephilidae in particular (e.g. Herennia, Fig. 1A, and especially Nephila, Fig. 1B) are becoming model organisms for the studies of extreme, femalebiased SSD and its consequences for sexual biology [9,10,11,12,13,14,15]. Previous studies have focused on the relative importance of selection for large female size versus selection for small male size [16] and the current phylogenetic evidence suggests that extreme SSD in orbweaving spiders, nephilids included, is almost always due to female gigantism rather than male dwarfism [5,16,17,18,19]. However, prior studies all focused on individual species or on supraspecific phylogenetic levels. Combined with the new species described here, a recent species level nephilid phylogeny [20] makes possible the most detailed analysis of size change in nephilids to date, and thus should enable more rigorous hypotheses about selective forces affecting SSD in spiders.
Nephila contains the largest web-spinning spiders (,10 cm leg span), which make the largest orb webs (.1 m diam.) [20,21]. Out of 150 available scientific names, only 15 Nephila species are valid [22]. Linnaeus described the first Nephila species in 1767 (now N. clavipes) and Karsch described the last genuinely new Nephila in 1879 (N. constricta); all more recent descriptions are synonyms. This paper reports the discovery of the first new Nephila species since 1879. The first specimen, a huge, distinctly different female collected in 1978 at Sodwana Bay, South Africa, was discovered in 2000 in the collections from Pretoria. Two expeditions specifically to find this species were unsuccessful, suggesting that perhaps the form was a hybrid or extinct. Then in 2003 a second, unmistakably conspecific specimen from Madagascar was discovered in a Viennese museum, thus weakening the hybrid hypothesis. Failure to find additional specimens in more than 2500 samples from 37 museums seemed to support the extinction hypothesis. However, two additional females and a male were recently collected in Tembe Elephant Park by South African colleagues, and it is now clear that N. komaci is a valid, new extant Nephila species.
Here, we provide a formal description of Nephila komaci sp. nov., add it to the existing nephilid phylogenetic matrix [20], reconstruct the evolution of mean female and male size, and test their correlation using phylogenetically independent contrasts.

Results and Discussion
The genus Nephila already contained the largest orbweaving spiders, but N. komaci now becomes the largest Nephila species known (Fig. 1C). Our phylogeny shows that nephilid female size increases monotonically (binomial test of ancestral Nephila nodes leading to N. komaci, n = 8, p = 0.004) and roughly sevenfold from implied ancestral values (Fig. 1C). This evolutionary trend is mainly due to Nephila; it alone is significantly larger than the family average or compared to any combination of the remaining genera (t test, n = 31, p = 0.017). The largest Nephila species all belong to one ''giant female'' clade, containing African species (e.g. N. komaci) and the Australasian N. antipodiana and N. pilipes (Fig. 1B). Throughout the family, females significantly more often increase in size rather than decrease at speciation events (binomial test of all paired ancestor-descendant nodes, n = 62, p = 0.049). Monte Carlo simulation shows that the ''giant female'' clade, except N. constricta, significantly exceeds expected body size (Fig. 1C, n = 15,000 replicates, p,0.05). However, nephilid male size oscillates within a threefold range (Fig. 1C), shows no significant trend with phylogeny, and is decoupled and independent from the evolution of female size (n = 26, r 2 = 0.055, p = 0.787). Monte Carlo simulation of male size, however, shows that males sporadically achieve significantly large sizes (Fig. 1C).
These species-level data reinforce Nephila sexual size dimorphism as female gigantism [5,16], rather than male dwarfism [18,19]. Large Nephila females may experience less predation [17] and, apparently at thresholds of roughly 28 mm body length, are freed to respond dramatically to fecundity selection for large size [17,23]. First male advantage, sperm competition, or climbing ability favor small size via early maturation, but direct male-male competition and female cannibalism of males favor large size [7,9,24,25]. Significant deviations from expected male size are all increases, suggesting that males do track females to some extent, but these increases are phylogenetically scattered (Fig. 1C). As a new member of the distal (giant) Nephila clade, N. komaci should be at the forefront of nephilid sexual size dimorphism research. If any other viable populations of this distinctive species exist they ought to be easy to locate (Fig. 2). Although the distribution data are currently scarce, the species may be threatened or endangered. It is nowhere abundant, the range is apparently restricted, and all known localities lie within two endangered biodiversity hotspots: Maputaland-Pondoland-Albany and Madagascar.  Nephilinae Simon 1894 sensu Kuntner [26] Nephila Leach 1815 Nephila komaci sp. nov. (Fig. 2 [27,28] failed to find N. komaci despite focused searches. Natural history/ecology: Mostly unknown, but see above. As with other Nephila species, N. komaci is predicted to spin a large golden orb web, with a three dimensional barrier web at least in early instars [20]. The two Tembe specimens were collected by beating a large shrub, thus the web was probably 2-4 m above the ground. Two other Nephila species (N. inaurata, N. fenestrata) are sympatric at Tembe. Conservation status: Nephila komaci is evidently rare (37 museum collections were examined in addition to field searches), and may be endangered because its only known habitat, Maputaland coastal forest is increasingly rare [29]. Diagnosis: Female N. komaci differ from all other African Nephila species except N. sumptuosa and N. inaurata by the shape of the abdomen, which is wide and long, and extends considerably beyond spinnerets ( Fig. 2A-B). Female N. komaci differ from those of N. sumptuosa by the ridged carapace edge ( Fig. 2A), the almost unicolorous sternum, and by lacking extensive fields of femoral short macrosetae. They differ from N. inaurata by a conspicuous yellow and brown abdominal dorsal pattern ( Fig. 2A-B) and the epigynum with slit-like copulatory openings (Fig. 2C-D). The male palp (Fig. 2E-F)  high, extended 4.9 beyond spinnerets. Dorsum (in ethanol) brown with a broad anterior yellow notched pattern, a mid-posterior paired and a caudal unpaired yellow patch; lateral opisthosoma brown with yellow spots and stripes; venter brown, with two irregularly shaped conspicuous yellow transverse bands. Epigynum a protruding sclerotized area and a posterior transverse plate with slit-like, medially converging copulatory openings (Fig. 2C-D). Round spermathecae juxtaposed medially. Copulatory ducts complex and long, fertilization ducts massive.

Methods
Taxonomic methods follow recent nephilid treatments [26,30,31], all measurements are in millimeters. Nephila komaci data added to a nephilid phylogeny [20] produced the same four topologies and preferred hypothesis (Fig. 1C). Although Fig. 1C depicts the evolution of mean female and male size (under squared change parsimony), all statistical tests used log (mean body length = average of minimum and maximum values) corrected via independent contrasts [32] using the PDAP module [33] in Mesquite [34]. We construed branch lengths as the count of unambiguous changes plus one (to correct for seven terminal zero length branches). For Monte Carlo simulations in Mesquite, we used an estimate of ancestral body sizes in nephilids (10.0 mm for females, 3.4 for males; linear parsimony reconstruction at the root), as the null hypothesis for body size under no selection. We adjusted the Brownian motion rate parameter so that for each sex the average simulated variance approximated the observed, and simulated body size evolution 15,000 times. SSD is defined as mean female body length: mean male body length. Extreme SSD is defined as SSD value exceeding 5. Using mean prosomal length as a measure of body size, or linear parsimony instead of squared, changes no statistical conclusions.

Nomenclatural Acts
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