SYSTEMATICS, TOXINS, PARASITOIDS, AND NICHE: AN
INTEGRATIVE APPROACH TO THE STUDY OF A NEW
BLACK WIDOW SPIDER IN COLOMBIA
Doctoral Thesis presented by:
Martha Alexandra Rueda Esteban
Advisor: Emilio Realpe PhD.
Co-Advisor: Adolfo Amézquita PhD.
Evaluator: Charles Griswold
Evaluator: Jorge Molina
Dissertation submitted in partial fulfillment of the requirements for the degree of Doctor in Philosophy Department of Biological Sciences Graduate School Universidad de los Andes Bogotá, Colombia. 2018
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Abstract
Black widow spiders have been widely studied because they are medically important due to the syndrome produced after a bite. The syndrome can produce local or systemic reactions and can be fatal. Also, because of the difficulty in their classification; the color pattern polymorphism, and the similarities in the reproductive systems have been a problem in the differentiation and taxonomic identification of species by morphology. There are 31 species of Latrodectus described worldwide, and two species described for Colombia, L. curacaviensis (Müller, 1776) and L. geometricus Koch, 1841.
L. geometricus is the only cosmopolitan species and has been classified as invasive in many ecosystems; this species origin is not known yet, but the closest phylogenetic relative is from Africa. L. curacaviensis was recognized as a species after many changes in its classification. This species was last reported in Colombia in the department of
Atlantico, north of the country.
Black widows in Colombia are known since 1538, when Gonzalo Jimenez de
Quesada reported deaths in his army by the bite of a deadly red spider in the “Valley of sadness”, the Tatacoa desert, southwestern Colombia. This unknown species has been described in many natural history essays and medical articles in Colombia since that date, but they refer to the spider with the common Latin-American black widow name “coya”; there is no reference to a specific species and the knowledge of the epidemiological problem this unknown species impose, is null.
According to previous studies, L. geometricus and L. curacaviensis have a Lethal
Dose 50 of 22.5 mg/kg and 12 mg/kg respectively, and the amount of dry venom injected in Latrodectus spiders ranges between 0.02-0.03 mg; showing that none of these two species can produce the symptoms described in the natural history essays and medical articles from the Tatacoa desert. The first chapter in this Thesis studies the medical
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importance of the unidentified species from the Tatacoa desert, in order to determine if that morphotype has the presence of the α-latrotoxin and can be the species reported in all the previous reports of Latrodetism in the Country. The toxin was found in the venom of the Latrodectus species found in the Tatacoa desert and the symptoms observed were consistent with the symptoms described for the syndrome.
Therefore, the species present in the Tatacoa desert do not belong to any of the species described for the Country and a study of the identity of the species, ecology, taxonomy, or evolution of this genus in the Country is needed. The second chapter identifies the Tatacoa spider as a new Latrodectus species, and describes a different morphotype in Santander, also classified as a new species. New population reports for 10 departments in the country are made for L. geometricus and L. curacaviensis. Also, a synonymy for two South-American species, described originally in Argentina, is determined. The species report is made by morphological and molecular data; based in the analysis of partial sequences of the mitochondrial cytochrome c oxidase subunit I
(COI), and the mitochondrial ribosomal RNA gene 16S (16SrRNA), including a phylogenetic and genetic analysis, that show enough support for the new species status.
One candidate species is described for Santander department, this species shows a close genetic relation to L. hesperus Chamberlin and Ivie, 1935, and the same color pattern change during ontogeny. The Tatacoa desert candidate species is described for Huila and
Tolima departments, and show a highly close genetic relation to L. corallinus Abalos,
1980 and L. diaguita Carcavallo, 1960; also, the genetic analyses show enough similarity between L. corallinus and L. diaguita to support the synonymy between these two species.
The Colombian population of the Tatacoa candidate species was found apparently co- existing with L. geometricus in the “desert”, until 2016 when it disappeared from the ecosystem and is presumed to be locally extinct.
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This local extinction raises the question about the possible co-existence, competition, and segregation of these two species in this community. The third chapter studies the two sympatric populations in the Tatacoa desert, and the possible variables that acted as ecological pressure in the local extinction of Latrodectus sp. The niche width was measured using several environmental and trophic variables to determine the overlap and evaluate the differences that allowed them to share the ecosystem for so many years.
The trophic variables used included dry cocoons of prey found in the webs analyzed by direct observation, and metagenomic data from gut content analysis. The environmental and trophic niche breadth of L. geometricus were bigger than the niche of Latrodectus sp. and a complete overlap was observed. After 2016, Latrodectus sp. was not found in the desert and a local extinction was declared. More populations of this species were found north of the desert, in dry and warm places where L. geometricus has not stablished yet.
A case of competition between two species is described, but the co-existence in a community is not only reduced to the inter-specific relations with just one species, ecological pressure given by predation and parasitism can also have influence in the population density of a species. Chapter four aims to expand the ecological analysis of co-existence and evaluate parasitoidism to determine if that could have been acting as ecological pressure and be one of the explanations to the local extinction of Latrodectus sp. in the Tatacoa desert. Two species of parasitoids were found in the egg sacs of both spider species while co-existing. Ecological variables were measured and habitat preference by the parasitoids was found. Also, number of healthy egg sacs and egg sacs with the presence of parasitoids by each species was determined. The sample number did not allow to evaluate host preference.
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Dedication
To my mentor and dear friend Emilio Realpe for teaching me the love for arthropods. To my co-advisor and guide Adolfo Amézquita.
To LAZOEA for all the support and help all these years.
To my family and friends, for being patient enough to survive the Ph.D. with me.
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Contents
Abstract ...... 2
Dedication ...... 5
General Introduction ...... 8
General Introduction References ...... 18
General Introduction Figure Legends ...... 27
General Introduction Figures ...... 28
Chapter 1: Toxicity evaluation and initial characterization of the venom of a Colombian
Latrodectus sp...... 30
Chapter 1 References ...... 44
Chapter 1 Figure Legends ...... 49
Chapter 1 Table Legends ...... 50
Chapter 1 Figures ...... 51
Chapter 1 Tables...... 53
Chapter 2: Two new species of Latrodectus (Araneae: Theridiidae) from the dry forests in the Magdalena Valley, Colombia ...... 55
Chapter 2 References ...... 74
Chapter 2 Figure Legends ...... 78
Chapter 2 Figures ...... 80
Supplementary Material Files: Chapter 2 ...... 88
Chapter 3: Niche overlap in two sympatric species of black widows in Colombia using ecological and genomic data ...... 111
Chapter 3 References ...... 131
Chapter 3 Figure Legends ...... 138
Chapter 3 Table Legends ...... 140
Chapter 3 Figures ...... 141
Chapter 3 Tables...... 145
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Supplementary Material Files: Chapter 3 ...... 146
Chapter 4: Parasitoid habitat preference in Colombian black widow spiders ...... 151
Chapter 4 References ...... 160
Chapter 4 Figure Legends ...... 164
Chapter 4 Figures ...... 165
General Conclusions ...... 167
Thesis Supplementary Material ...... 169
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General Introduction
Colombia is a Megadiverse Country that has a great variety of ecosystems, most of them threatened by human action to extinction (Pizano et al. 2014). Dry forests are one of the most endangered ecosystems in the country, with only 8% left; the soils are relatively fertile, and they have been used for agriculture, livestock, mining, urban development, and tourism; having a great effect on the associated biodiversity (Pizano et al. 2014). 65% of the original dry forests of the country are desertified by the unsustainable use, they are distributed in five regions of the Country: Caribe, Orinoquía,
Valle del Cauca, Magdalena basin, and Patía Valley in Nariño (Pizano et al. 2014). There is a high endemism in these regions because of the specific adaptations the organisms have developed to survive dry periods (Pizano et al. 2014). The Biodiversity studies in dry forests in Colombia are centered in plants (Figueroa and Galeano 2007), amphibians
(Acosta-Galvis 2012), flies (De Polanco et al. 2008), scorpions (Gómez and Otero 2007;
De Armas et al. 2012) and spiders (Rueda et al. 2017).
The studies in spiders have increased worldwide, there are 46.211 species described to date, and they can reach 170.000 according to estimates (Platnick 2017). The study of spiders in Colombia is mainly centered in tarantulas, due to illegal traffic and their beautiful coloration (Amat-García et al. 2007). In Colombia there are 914 species of spiders described to date and in the dry forests, the records are very low; studies include spiders from the Families Theriididae, Salticidae, Lycosidae, Oxyopidae, and several
Mygalomorphs (Amat-García et al. 2007).
Theridiidae family is the second biggest family among the spiders, after
Salticidae, and they show a world wide distribution (Kaston 1970). These spiders show a great variety in morphology, ecology and behavior; also, they show a great diversification in the web structure that gives an idea of the phylogenetic location of interior taxa (Foelix
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2010). One of the most studied genus inside Theridiidae family is Latrodectus, commonly called black widow spiders (Agnarsson 2004); they are considered dangerous by the
World Health Organization (Quintana and Otero 2002), because of the severe clinical manifestations and possible lethality of the venom (Bonnet 2004; Aguilera et al. 2009;
Calvete et al. 2009).
Latrodectus venom contains peptides or protein fractions (Boevé et al. 1995) that act as toxins affecting the central nervous system of different organisms; these include latrotoxins and trigger a massive neurotransmitter release upon injection (Shukla and
Broome 2007; Aguilera et al. 2009; Garb and Hayashi 2013). One of the venom components, the α-Latrotoxin, is toxic to mammals. The bite of some species like
Latrodectus mactans (Fabricius, 1775) can be lethal (Quintana and Otero 2002; Rohou et al. 2007) but few cases are lethal if treatment is supplied (Artaza et al. 1982). The first symptoms that occur are local pain, sweating, itching, and edema. Systemic symptoms include fever, dizziness, cramps, spasms, dyspnea, tachycardia, arrhythmia, among others
(Duan et al. 2006; Guerrero et al. 2010). These symptoms can last a few hours to more than a week and can lead to death because of pulmonary or cerebral edema or cardiovascular manifestations (Quintana and Otero 2002). The symptoms also depend on the spider species, there are reports of differences in severity of the effect of the bite between species and among close related genera, like Steatoda and Parasteatoda (Garb and Hayashi 2013). Also, it has been proved that spiders can modulate the amount and concentration of the venom for eating and defense (Nelsen et al. 2014). The median lethal dose in mice is already known for several species of Latrodectus (Daly et al. 2001; Reyes-
Lugo et al. 2009).
Latrodectus genus has a worldwide distribution (Figure 1), and there are 31 species described to date (Platnick 2017). The genus forms a monophylogenetic group,
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but the internal relations of the species have been a challenge; there are no clear morphological boundaries between them (Levi 1983). These spiders show a low variation in characters such as coloration, color pattern, setae presence and abundance, and genitalia; characters that are diagnostic in the identification and species delimitation in other taxa (Levi 1959; 1983). DNA barcoding has become an important tool for spider identification, specially inside Latrodectus (Garb et al. 2004) and Cytochrome oxidase subunit I (COI) has proven to be effective solving phylogenies for this group (Garb et al.
2004; Barrett and Hebert 2005).
Black widow spiders have a globular abdomen and small body size (Quintana and
Otero 2002). They tend to be calmed, not aggressive, but they can attack when compressed or bothered (Grisolia et al. 1992). All spiders have specific high molecular toxins in the venom that affect the central nervous system, muscular system or show circulatory affections (Boevé et al. 1995; Ushkaryov et al. 2004), but only few species can produce a severe syndrome in mammals (Lelianova et al. 1997; Ushkaryov 2002).
The syndrome produced after a bite is called Latrodectism and it is rarerly fatal; the common symptoms are severe muscular pain, spasms, nauseas and diaphoresis (Quintana and Otero 2002; Haney et al. 2014). The spiders are diurnal, and active during summer time, they have sexual dimorphism with the female bigger than the male (Herzig and
Hodgson 2009).
In Colombia, the research of the genus is new, and the information available for epidemiology studies is rare; additionally, the accidents by venomous spiders are not of mandatory notification to the National Health Institute (INS). There are two species of
Latrodectus described in for the country: L. curacaviensis (Müller, 1776) and L. geometricus Koch, 1841. There have been reports of Latrodectism in Tolima and
Atlantico, but the spider identity is never mentioned (Quintana and Otero 2002). There
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are several studies in the country about Latrodectus, but they mention it with the common name “coya”. The first scientific records, where the medical importance is named are from 1914 and 1938 (Aguirre-Plata 1914; Hamburger 1938). Later, they are mentioned in the “Essay of American History”, where priest Felipe Salvador Gilij explains that he found a red spider in the Tatacoa desert, not aggressive, but with a very strong venom that produces “deadly spasms” when compressed with bare hands (Gilij 1955). In 1985, the French naturalist Boussingault, wrote that the red spider is still an enigma, because people fear it as “the most dangerous animal they can find”, but the accidents reported are not as common as they should be (Boussingault 1985).
L. geometricus (Figure 2) was described by Koch in 1841, the type specimen was collected in the Tatacoa desert in Colombia (Vincent et al. 2008; Marie and Vetter 2015).
It is the only black widow spider with a cosmopolitan distribution, and is considered invasive in many ecosystems. The wide distribution is owed to human transport that has increased the geographical range, and to the great adaptability and affinity to disturbed ecosystems (Vetter and Isbister 2008; Lewis 2013). There are reports of populations in
Hawaii, California, Jamaica, Bermuda, Haiti, Cuba, Israel, Turkey, India, Australia,
Papua New Guinei, Indonesia, Filipins, Japan, and South America, but the clossest phylogenetic relative is African (Garb et al. 2004; Vetter et al. 2012; Marie and Vetter
2015). This species builds a small funnel in a corner of the web, and the egg sac has a characteristic pointy surface (Figure 2) (Vetter et al. 2012). It also shows low genetic divergence between populations (Marie and Vetter 2015).
L. curacaviensis (Figure 3) was described by Müller in 1776, with the type specimen from Curacao (Müller, 1776). The caparace shows a light brown coloration, deeper in the thoracic depression and sternum. The legs show a dark brown color with a darker portion in the joints. The abdomen shows a black background with red shapes
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(McCrone and Levi 1964). The genitalia is very similar to other described species as L. variolus Walckenaer, 1837 and L. bishopi Kaston, 1938, but the color patterns are different. It has not been found again in the type locality, maybe because of ecosystem modification by cattle raising and desertification, and it seems to be displaced by an invasive population of L. geometricus (McCrone and Levi 1964). The natural history, behavior and evolution history has not been studied, and the actual distribution in
Colombia is unkown.
Toxicity studies of several invertebrates made by Bucherl in 1971, show that the mean lethal dose for L. geometricus is 22.5 mg/kg, and for L. curacaviensis is 12 mg/kg; the maximum dose extracted for each species was of 0.5 mg and 1.3 mg respectively
(Bucherl and Buckley 1971) and the average amount of dry venom injected in
Latrodectus spiders ranges between 0.02-0.03 mg (Maretić 1987). With these data, it can be infered that none of the species reported for the country are capable of producing a severe syndrome in a human adult, like the syndromes described in medical articles
(Aguirre-Plata 1914; Hamburger 1938) of the black widow found in the Tatacoa desert, so the identity of the red spider named in the scientific reports and assays is still unknown.
A red Latrodectus morphotype was found in the Tatacoa desert, consistent with the geographic location and morphological description of the “coya” spider. In order to evaluate if this morphotype could be the spider named in all historical records, a toxicity evaluation and initial characterization of the venom of this species was performed in chapter one of this investigation. The study aimed to evaluate the toxicity of the venom with mice models and compare the symptomatology of the Latrodectism syndrome produced by the bite of other reported species in Colombian species to other reported species. This study is the first approximation to the extraction and venom analysis for a species of this genus in the country and it is important to determine whether this species
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can become a health problem in the areas near these spider populations. The study showed the presence of the α-latrotoxin in the venom of this unknown Colombian Latrodectus species; also, all animal models died during the study and showed symptoms consistent with Latrodectism. The results support the hypothesis that this species is the one described in medical articles and natural history essays, but the identity is still unknown. Little is known about the natural history, ecology and biology of this genus, the absence of statistical data about accidents by its bite and the profound fear people have, gives enough reasons to study and determine new population reports, and new species for Colombia.
In chapter two, we develop a phylogenetic hyphothesis about the study case in the
Tatacoa desert, and aims to find more species or populations reports for the country. This study aims to increase the knowledge of this genus in Colombia and understand the genetic relation between all populations and species related with the identified taxa. A dense sampling across the country was accomplished, and also, we received samples from scientists from different localities. Molecular markers such as the mitochondrial cytochrome c oxidase subunit I (COI), and the mitochondrial ribosomal RNA gene 16S
(16SrRNA), and morphological characters, were used to identify the sampled individuals.
Two candidate species are identified; the unknown morphotype recovered in the Tatacoa desert, and a morphotype collected in the department of Santander, also, the synonimy of
L. corallinus and L. diaguita is proposed. All hypothesis are supported by genetic and morphological analyses. New population reports are made for L. geometricus and L. curacaviensis, and sympatry between Latrodectus species is recorded as a common phenomena in Colombia, with the first case being reported in the Tatacoa desert.
In 1841, Koch described L. geometricus with a type specimen collected from the
Tatacoa desert (Koch 1841; Vincent et al. 2008; Marie and Vetter 2015), and historical records place the red “coya” spider in the Tatacoa desert since 1538, when Gonzalo
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Jimenez de Quesada reported deaths in his army by the bite of a deadly red spider
(Aguado 1916). After that it has been named in many natural history essays and medical articles in Colombia since that date (Aguirre-Plata 1914; Hamburger 1938; Gilij 1955;
Boussingault 1985). Our investigations give enough support to the hypothesis of the co- existence of L. geometricus and a candidate Latrodectus species for more than 175 years in this specific ecosystem (Gilij 1955; Boussingault 1985; Marie and Vetter 2015). After
2016, Latrodectus sp. could not be found in the Tatacoa desert, despite intensive search, and is presumed to be locally extinct. Potential causes of this extinction are evaluated. In chapter three, the hypothesis of co-existence, competition, and niche overlap was studied in order to evaluate if L. geometricus was acting as an invasive species in the Tatacoa desert and determine if segregation of Latrodectus sp. happened, during the time sympatry took place. This segregation phenomena by L. geometricus has been observed in different ecosystems (McCrone and Levi 1964; Marie and Vetter 2015).
Sympatry in a community implies co-exitence or competition, and can be explained by the analysis of ecological disturbance or niche differentiation; trough the study of interaction networks or food webs (Bulleri et al. 2016). These species interactions are important to understand community dynamics (Spiller and Schoener
1994). The species interactions can be described in terms of different variables related to the micro-habitat, such as temperatures, humidity, food, etc. And they can show specialization, in highly biodiverse ecosystem where resources are shared between different lineages. Niche is defined as “The fitness measure of a species in a multi- dimensional environment” (Holt 1987) and can be characterized as the utilization measures of a resource (Pianka 1974); they have been used to understand species interaction and define the structure of a community. Sympatric species can make habitat partitioning in order to avoid competition when they use the same resource, this way they
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avoid overlap (Hardin 1960; Holt 1987). This partitioning can be observed in changes of behavior in a prey-predator system.
The interaction network can de described thought the study of ecology in each lineage. It plays an important role in speciation because incipient species occur in different environments or use resources in a different way (Wiens 2004). Natural selection on ecologically important traits can lead to different adaptations, evolutionary divergence, and reproductive isolation (Wiens 2004). Species tend to retain similar ecological niches over evolutionary time, and they fail to adapt to new environmental conditions; this is the “key factor” in initially isolating populations and the creation of new lineages (Wiens 2004). According to Wiens in 2004, during the dispersal process, the ancestral species has an ecological niche defined; when the new population is formed in an isolated geographic area with similar ecological conditions, niche conservationism determine the locations that are acceptable for colonization (Wiens 2004). The traditional view assumes that natural selection, adaptation and ecological specialization are important in the divergence of related lineages when dispersal takes place (Wiens 2004).
In both views, ecological specialization limits the habitat breadth of a species (Gruner
2007). It is important to determine and analyze the ecological niche variations between the ancestral and divergent populations, to understand the formation of different species or co-existence in a community. One of the ways of doing it is measuring the ecological breadth of each lineage in each population. This can be done by ecological measurements and analysis of the trophic differences.
In order to study the sympatry case in the Tatacoa desert between two species of black widow spiders, we used 8 micro-habitat variables: Environmental temperature, relative humidity, internal and external substrate temperature, length and width of the web, spider temperature and body length. Also, prey composition was evaluated for both
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species by comparing the recovery of prey taxa at different taxonomic ranks (Order,
Family, and genus), and comparing the diversity estimates of the recovered prey communities for each predator species. Empty prey cocoons, and molecular gut-content analysis were used for evaluating feeding niche differentiation. This is the first approach in the determination of niche breadth, and natural diet of black widows in Colombia, and the first time molecular techniques are used for trophic niche in this genus. We recovered similarities in environmental variables, and in prey composition, showing complete niche overlap between these two species.
Also, L. geometricus showed higher reproductive rates, that increases the success and dispersion speed; this species can produce an egg sac every third day and has an average of 18 to 30 egg sacs in the life span (Marie and Vetter 2015). If this reproductive rate is maintained, it can become a significative invasive species unless it is controled by an agent like parasites, parasitoids or predators to control the population density (Marie and Vetter 2015). Furthermore, inter-specific relations in a community includes relations with parasites and predators that control population densities and allow co-existence of species. We observed parasitoids in the egg sacs of both sympatric species, chapter four is the first study of behavioral responses in Colombian Latrodectus wasp parasitoids, and aims to identify the different wasp parasitoid species found in the egg sacs of the two sympatric populations of Latrodectus in the Tatacoa desert, determine if there is any difference between the egg sacs with the presence of the parasitoid and the healthy ones, and evaluate if these parasitoid wasps show habitat preference measuring environmental temperature, relative humidity and substrate temperature.
There are several insects that have been identified and used as biological control against invasive populations of Latrodectus, like a fly: Pseudogaurax signatus, a mantispid: Zeugomantispa minuta, and wasps: Philolema latrodecti and Baeus latrodecti
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(Bianchi 1945; Brambila and Evans 2001; Bibbs and Buss 2011; Vetter et al. 2012); most of these were known to affect L. geometricus egg sacs (Marie and Vetter 2015).
Latrodectus parasitoids have been described since 1942 in L. hesperus (Bianchi 1945), and in Latin America Pediobius pyrgo has been described as parasitoid in egg sacs of L. geometricus (Schoeninger et al. 2015).
Parasitoids need a suitable environment, after finding it, the search for the host starts (Vinson 1976). The habitat preference can be influenced by temperature, humidity, light intensity, wind, and habits of the parasitoids (Vinson 1976). There is extensive research in chemical cues that parasitoids use to find the host, but the physical factors, like humidity and temperature, have not been deeply studied in wasp parasitoids (Steidle and Van Loon 2003).
Two parasitoid wasps were identified for colombian species of Latrodectus; the results are shown in chapter four of this thesis. Philolema latrodecti and Pediobius sp., wasps are described, and they are more often encountered in microhabitats with high relative humidity and low temperatures. According to this, parasitoids can be a measure of ecological pressure depending on the thermal preferences by each spider species; rather than showing the cues used by parasitoids to detect host microhabitats, our data show the conditions that render spider egg sacs more vulnerable to attack by parasitoids.
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General Introduction Figure legends
Figure 1. Distribution of Latrodectus genus. Each point is in the center of the known distribution for each species. an, L. antheratus; ap, L. apicalis; at, L. atritus; bi, L. bishopi; ci, L. cinctus; co, L. corallinus; cu, L. curacaviensis; da, L. dahli; di, L. diaguita; er, L. erythromelas; ha, L. hasselti; he, L. hesperus; hy, L. hystrix; in, L. indistinctus; ka, L. karooensis; kt, L. katipo, li, L. lilianae; ma, L. mactans; me, L. menavodi; mi, L. mirabilis; ob, L. obscurior; pa, L. pallidus; qu, L. quartus; re, L. renivulvatus; rv, L. revivensis; rh,
L. rhodesiensis; tr, L. tredecimguttatus; vg, L. variegatus; and va, L. variolus. L. geometricus distribution in located with solid circles, modified from: (Garb et al. 2004).
Figure 2. Left: L. geometricus web funnel and characteristic pointy egg sac; taken from:
(Vetter et al. 2012). Right: L. geometricus female, picture by: Alexandra Rueda, 2014.
Figure 3. L. curacaviensis female, picture by Yiselle Cano, 2015.
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General Introduction Figures
Figure 1.
Figure 2.
Figure 3.
29
Chapter 1:
Toxicity evaluation and initial characterization of the venom of a
Colombian Latrodectus sp.
ALEXANDRA RUEDA1*, EMILIO REALPE1, ALFREDO URIBE2.
Corresponding authors:
Alexandra Rueda ([email protected])
Running Title:
Venom characterization of a Colombian Latrodectus
Cited references: 36
Figures: 4
Scope: Latrodectus toxicity evaluation
Published in Journal: Toxicon
Rueda, A., E. Realpe, et al. (2017). "Toxicity evaluation and initial characterization of
the venom of a Colombian Latrodectus sp." Toxicon 125(1): 53-58.
30
Toxicity evaluation and initial characterization of the venom of a
Colombian Latrodectus sp.
ALEXANDRA RUEDA1*, EMILIO REALPE1, ALFREDO URIBE2.
1 Laboratory of Zoology and Aquatic Entomology, Department of Biological Sciences,
Universidad de los Andes, AA 4976. Bogotá, Colombia.
2 Biochemical Investigation Center CIBI, Department of Biological Sciences,
Universidad de los Andes, AA 4976. Bogotá, Colombia.
*Corresponding author: [email protected]
Abstract
The genus Latrodectus has not been studied in Colombia even though it is medically important worldwide; there are three species for the country, this study focused on a non- identified species found in the Tatacoa Desert in the Huila Department. This research is the first approximation to the extraction, composition analysis and toxicity evaluation of the venom of a species of the genus Latrodectus in Colombia; and aims to evaluate the toxicity by the initial characterization of its venom. The venom extraction was accomplished with electrostimulation and total protein concentration was determined by the Lowry method and BCA assays from crude venom; with these methods, high protein concentration of the samples was measured. Bioassays on mice were also made to evaluate the toxicity and compare the symptoms produced by this Colombian spider to the Latrodectism Syndrome. Finally, an SDS-PAGE electrophoresis was used to separate the main components of high molecular weight from the samples and compared to a
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control of the venom of Latrodectus mactans to determine if the venom composition is different between these two species. α-latrotoxin was identified in the venom of this
Colombian species; also, the symptoms observed were consistent with the symptoms described for the latrodectism syndrome.
Key words: Spider, SDS-PAGE, black widow, Latrodectism.
Resumen
El género Latrodectus no se ha estudiado en Colombia, aunque es reconocida medicamente a nivel mundial; hay tres especies para el país. Este estudio se centra en una especie no identificada que se encuentra en el desierto de la Tatacoa, Departamento del
Huila. Esta investigación es la primera aproximación a la extracción, análisis de composición y evaluación de toxicidad del veneno de una especie de Latrodectus en
Colombia; y busca evaluar la toxicidad y realizar una caracterización inicial de su veneno.
La extracción del veneno se llevó a cabo por electro- estimulación, y la concentración de proteína total en la muestra de veneno crudo se calculó por medio del método Lowry y el análisis por BCA; con estos métodos se obtuvo una alta concentración de proteína en las muestras. También se realizaron bioensayos en ratones para evaluar la toxicidad y comparar los síntomas producidos por esta especie colombiana, con los síntomas relacionados con el síndrome del latrodectismo. Finalmente se realizó una electroforesis
SDS-PAGE para separar los principales componentes de alto peso molecular y compararlos con una muestra control del veneno de Latrodectus mactans y determinar si la composición entre las dos especies es diferente. Se identificó la α-latrotoxin en el veneno de esta especie colombiana, y los síntomas observados son consistentes con los descritos para el síndrome del latrodectismo
Palabras clave: Araña, SDS-PAGE, viuda negra, latrodectismo
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Short summary
The first approximation to the extraction, composition analysis and toxicity evaluation of the venom of a species of the genus Latrodectus in Colombia.
Introduction
Latrodectus (Walckenaer, 1805) is a spider genus that belongs to Family
Theridiidae (Sundeval, 1833). These spiders have a globular abdomen, small body size, sexual dimorphism and show a wide color pattern polymorphism in the abdomen, between and inside species. They live in dry habitats or near caves where the webs can be found (Quintana and Otero 2002; Garb et al. 2004). They are extremely calm, but they tend to bite when they are bothered or compressed (Artaza et al. 1982; Grisolia et al.
1992). The World Health Organization considers this genus dangerous (Quintana and
Otero 2002), and medically important because of the severe clinical manifestations and possible lethality of the venom (Bonnet 2004; Aguilera et al. 2009; Calvete et al. 2009).
The venom contains peptides or protein fractions (Boevé et al. 1995) that act as toxins affecting the central nervous system of different organisms; these include latrotoxins and trigger a massive neurotransmitter release upon injection (Shukla and Broome 2007;
Aguilera et al. 2009; Garb and Hayashi 2013). One of the venom components, the α-
Latrotoxin, is toxic to mammals. The bite of some species like Latrodectus mactans can be lethal (Quintana and Otero 2002; Rohou et al. 2007) but few cases are lethal if treatment is supplied (Artaza et al. 1982). The symptoms presented after the bite of a black widow or close relatives like Steatoda is known as the latrodectism syndrome
(Haney et al. 2014). The first symptoms that occur are: local pain, sweating, itching and edema. Systemic symptoms include fever, dizziness, cramps, spasms, dyspnea, tachycardia, arrhythmia, among others (Duan et al. 2006; Guerrero et al. 2010). These
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symptoms can last a few hours to more than a week and can lead to death because of pulmonary or cerebral edema or cardiovascular manifestations (Quintana and Otero
2002). The symptoms also depend on the spider species, there are reports of differences on severity in the effect of the bite between species and among close related genera, like
Steatoda and Crustulina (Garb and Hayashi 2013). The median lethal dose in mice is already known for several species (Daly et al. 2001; Reyes-Lugo et al. 2009), these rise the question of whether this Colombian species can be medically important.
There are three species described in Colombia: L. curacaviensis (Müller, 1776) and L. geometricus Koch, 1841 (Quintana and Otero 2002) and an un-identified species that is found in the Tatacoa Desert, located in the State of Huila (Rueda 2010). This last species has a different abdomen coloration pattern and is found on different clades in a molecular reconstruction (Rueda 2010). Also, there have been cases of latrodectism reported for L. geometricus and L. curacaviensis in the states of Tolima and Atlántico
(Quintana and Otero 2002). In Colombia the research for the genus is new and the information available for epidemiology studies is rare; additionally, the accidents by venomous spiders are not of mandatory notification to the National Health Institute (INS).
This study is the first approximation to the extraction and venom analysis for this genus in Colombia, with the aim of evaluating the toxicity of this species venom and compare the symptomatology to the latrodectism syndrome. It is important to determine whether the species present in Colombia can become a health problem in the areas near the spider populations.
Materials and Methods
Specimen recollection: The habitat of this species is the Tatacoa Desert, a dry forest located 440 meters above sea level in southwest Colombia, Huila State, but the real
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distribution is not yet known. The webs rarely are above 50 cm from the ground and they are usually anchored to rocks or small dry forest plants (Rueda 2010). The collection was made during the day and the spiders were kept in individual jars with a small receptacle for water (See Figure 1). They were taken to Bogotá and maintained at 29°C in the
Zoology and Aquatic Ecology Laboratory LAZOEA at Universidad de los Andes, in small rounded acetate cages of 15 cm of diameter. The photoperiod was controlled, and the specimens received 12 h of light. The spiders were fed with common crickets (Acheta domestica) every three weeks according to protocols stablished (Horni et al. 2001;
Zschokke and Herberstein 2005). The spiders were identified as Latrodectus by morphological determination and molecular analysis using COI as genetic marker (Rueda
2010).
Venom extraction: After the specimens were fed, they were left for minimum two weeks with just water before the venom extraction protocol in order to avoid contamination with gut contents, the extraction protocol for widow spiders is not easy due to their small size (Rocha-e-Silva et al. 2009). The spiders were sedated with CO2 before being handled (Smith 2015), then they were immobilized by the posterior portion of the cephalotorax, near the pedicel with a modified forceps for this procedure (See
Figure 2).
Once the specimen is immobilized, the body of the spider is moistened with saline solution and the cepaholotorax covered with two copper cables, one dorsal and one ventral portion of the body near the chelicerae. The spider was then stimulated by electric shocks of 40V with intervals of 1 to 2 seconds and for no more than 5 seconds. The venom was collected with modified glass capillary and then frozen at -20°C. This process was carried out once a month with each specimen, two to three weeks after the feeding took
35
time. This electro-stimulation protocol was modified from published versions used for different arthropods (Meadows and Russel 1970; Rocha-e-Silva et al. 2009; Garb 2014).
Total Protein Quantification by Folin-Lowry Protocol: The method was modified from manufacturer’s protocol, in order to use minimum sample sizes. For calibration curve, 5 solutions of bovine serum albumin (BSA) were used with known concentrations of 25, 50, 100, 200 and 400 mg/dl. The test was accomplished with two calibration curves and two replicates of crude venom each one with 2 µl final volume and was read at 620 nm (Lowry et al. 1951).
Total Protein Quantification by Bicinchoninic Acid (BCA): This colorimetric method allows the quantitative determination of the total protein concentration on a sample, it is more sensitive and faster than Folin-Lowry (Bainor et al. 2011); and was used as a standardization and validation of the previous results. Five solutions were used for the calibration curve with bovine serum albumin (BSA) with known concentrations of 25, 50, 100, 200 and 400 mg/dl; and two replicates of crude venom were analyzed each one with 2 µl of volume. The tests were performed using the Thermo Scientific Pierce
Kit (BCA Protein assay kit) according to manufacturer’s protocol and was read at 562 nm
(Bainor et al. 2011).
Bioassays in mice: Groups were formed depending on the weight and age of each mouse, two replicates were used per group and each group had its control (See Table 1).
The mice were sedated with Ether (Carneiro et al. 2002) and then immobilized, they were bitten by an adult female Latrodectus in the leg or the base of the tail (Gomes et al. 2002;
Rodrigues et al. 2004). The bite must be seen by the researcher in order to consider it a valid experiment. The symptoms and timeline were analyzed. (All experiments were approved by the Ethics Committee from Universidad de los Andes (CICUAL) and followed the normativity stablished by the “Guide for the care and use of laboratory
36
animals” from the National Research Council of the National Academies (Barthold et al.
2011)).
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE):
In order to determine the protein components of different venoms, many scientists have used this technique because of its high specificity and easiness separating components by molecular weight (De Lima et al. 2000; Barona et al. 2004; Duan et al. 2006; Liu et al.
2009). For the electrophoresis, a two density acrylamide gel was used, the upper layer at a density of 5% and the lower at 12% to allow a better high molecular protein separation.
A vertical electrophoresis was made in reduced conditions at 125 V for 90 minutes. The molecular weight markers used were: Fermentas Page Ruler Plus Prestained Protein
Ladder #SM1811 and Sigma Molecular Weight Marker SDS7. The loading buffer was a modified Coomasie G-256 recipe.
Three different crude venom concentrations were run, according to previous standardization with the venom of a Colombian Bothrops asper (Venom obtained from a previous CIBI investigation): 1, 3 and 5 µl of total protein, a sample of the crude venom of Latrodectus mactans obtained by Spider Pharm (Spider_Pharm 2014) was placed in the last two wells; as a concentration control the venom of Bothrops asper was placed on the wells 3 and 4. Then the gel was stained with Coomasie Blue for one hour, after reading it, the gel was discolored in a solution of acetic acid, methanol and water to perform a silver stain with the Bio-Rad Silver Stain Plus #161-0449. The bands were observed in a transilluminator for both colorations.
Results
In total, 288 specimens were collected during two years, sampling two times each year. The extraction protocol by electric shocks allowed maintaining the specimens alive
37
for a longer period of time, after some time the spiders got used to the electric stimulus, so the Voltage had to be increased gradually.
For the total protein quantification using the Folin-Lowry test, the mean values of the calibrating curve were averaged with the analytic values of 20 calibration curves of the Investigation Center in Biochemistry CIBI from Universidad de los Andes. The lowest correlation coefficient obtained from the standards between absorbance and known concentration was of 0.991. This indicates that those values can be used to calculate the concentration of protein from the crude venom. The mean concentration obtained by this protocol was 1284.5 mg/dl. For the total protein quantification using the
Bicinchoninic Acid (BCA) method, the mean values of the calibrating curve were averaged with the analytic values of 50 calibration curves of the Investigation Center in
Biochemistry CIBI from Universidad de los Andes. The lowest correlation coefficient obtained from the standards between absorbance and known concentration was of 0.995.
The mean concentration obtained was of 1015.1 mg/dl.
In the bioassays with mice, all experimental models died, the symptoms observed are shown in Table 2 separated by groups, visible symptomatology, and mean time between the appearances of each symptom among replicates are shown.
To standardize the minimum sample concentration needed to see a clear band in the
Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) that was performed, snake venom was used (Bothrops asper) for preliminary analysis. The minimum concentration to see a band was of 1.5 µg of protein and the best results were yielded with a concentration of 2.5 µg of protein in the sample. After these volumes were determined, the SDS-PAGE was performed with the extracted Latrodectus sp. venom.
After the Coomasie coloration a picture was taken using the transilluminator and saved
38
for comparison with the second coloration method. The same gel was used for both colorations.
After the Coomasie coloration the gel was washed and cut, eliminating the last two wells to improve quality. It was then placed under silver coloration that is more sensitive and a picture was taken with the transilluminator (See Figure 4). The three different concentrations of Latrodectus sp. venom (wells 3, 4 and 5) showed the same results, but the better bands were observed in well 5, where the crude venom concentration was of 1.015 µg of protein. In both coloration methods a separation of the high molecular weight protein component was accomplished, allowing the lower weight components to flow out of the gel. A faint band is observed around the weight of 130 kDa, that corresponds to the main latrotoxins and where the α-latrotoxin is located. The venom of L. mactans (wells 9 and 10) showed a similar band pattern above 70 kDa between the two species in the gel. The concentration of protein from L. mactans was higher resulting in a heavy band and transfer to the next empty well. The band pattern observed for medium and low weight proteins are different between L. mactans and the
Colombian species as seen in the band pattern below the 70 kDa band, it can be seen in
Figure 3 where the bands present in the Colombian species studied in this investigation are marked and compared. From the results of the electrophoresis we can calculate a correlation between the logarithm of the molecular weight (MW) of each marker versus the migrated distance of each band. There is a negative correlation between the distance migrated and the logarithms of the MW, as expected, and shows a R2 of 0.9646. In order to calculate the molecular weights of each band from the sample protein we used just one curve with the values of the two markers together obtaining one regression equation.
With this equation we were able to calculate some of the protein molecular weights present in the crude venom sample of Latrodectus sp. (See Table 3 and Figure 3).
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Discussion
Latrodectus spiders have been widely studied because of the medical importance, due to the affinity of the toxin for the cells from the nervous system in mammals; and because they produce serious symptomatology in humans (Artaza et al. 1982; Quintana and Otero 2002; Bonnet 2004). That is why the main objective of the present investigation was to evaluate the toxicity of the venom of a Colombian widow spider, and to evaluate the effect produced on mammal cells of different weights; in order to determine if this species could have medical importance.
There is little information about the venom extraction protocols by electric shocks in widow spiders, and because of their reduced size and lack of aggressiveness it is very difficult to obtain; but it has been described in a video step by step by Garb in 2014
(Rocha-e-Silva et al. 2009; Garb 2014). The extraction protocol described in the video was modified so the specimens would not habituate to the stimulus, an effect we found after several experiments with the same specimen. The capillary accomplished a double function: recovering the venom sample, and mechanically stimulating the chelicerae to ease the release of the venom. With this protocol we were able to obtain enough venom to accomplish the investigation without sacrificing any specimen. It is important to consider that with the electric shock protocol the venom can be easily contaminated by enzymes from the digestive fluid, but it can still be a clear colorless fluid (Herzig and
Hodgson 2009; Liu et al. 2009), that is why the most used protocol for venom extraction is the dissection of the venom glands (Duan et al. 2006) but this implies the death of all the spiders used in the study.
The bioassay in mice proved that the spider's venom has an effect on the nervous system of the mammals that is consistent with the presence of the presence of latrotoxin neurotoxins in the venom of this species of Latrodectus. The symptoms produced after
40
the bite were consistent with the symptoms described in the Latrodectism syndrome
(Quintana and Otero 2002; Bonnet 2004; Duan et al. 2006; Guerrero et al. 2010). The symptomatic triad was observed in the groups where the mice where older and bigger:
Muscular pain, Diaphoresis and psychomotor excitement (Artaza et al. 1982). In the first group, where the mice were newborn, the only observable symptoms were spasms, redness in the bite site and erratic movement. The symptoms were measured since the bite was observed; the most visible were diaphoresis, excessive salivation, spasms and breathing difficulty. These results confirm that this spider could be a potential danger to the local communities near the Tatacoa Desert due to the venom effect on mammals; the symptoms observed could be consistent to the presence of the α-Latrotoxin, but in order to prove that, more experiments should be made. With the aim of confirmation, the presence, concentration and the LD50 of the α-Latrotoxin must be determined.
Latrodectus venoms contain different kinds of toxins (neurotoxins, assistant toxins, proteases, protease inhibitors and unknown function toxins) and there is evidence that these different toxins can work together by binding to the target cells, enhancing the toxicity, protecting toxins and cleaving precursors to get mature toxins (He et al. 2013).
The toxins of the Latrodectus genus are known to be proteins of high molecular weight
(Rash and Hodgson 2002; Ushkaryov et al. 2004; Liu et al. 2009; Haney et al. 2014) called Latrotoxins that have a molecular weight between 110 and 140 kDa (Rash and
Hodgson 2002; Bonnet 2004; Duan et al. 2006). This explains why the protein concentration obtained in the experiments was high, because the venom of black widows is a complex mixture of toxins with different functions (He et al. 2013). This complexity makes the production of the venom an expensive process for the spider, and there are reports of control over the amount and production of dry bites; a process called venom optimization, an active control of the amount of venom used in defense and predation
41
(Nelsen et al. 2014). It is important to notice that this research used direct bites in the experiments, so the results are not showing variations in the amount of venom injected to the mouse in each bite.
In the polyacrylamide electrophoresis performed, the lower molecular weight components were allowed to flow out from the gel until the high molecular weight components were separated. According to the results it can be seen that the Coomasie
Coloration has a better resolution than the silver coloration and allows a better analysis of the protein bands. There are visible bands at 170 kDa, then at 130 kDa a protein in low concentration and then another band above the 100 kDa. The black widows are characterized for presenting several high molecular weight toxins that encode for different functions (Haney et al. 2014) and this bands should be characterized to identify the exact component present in the venom. The concentration of the crude venom of L. mactans is very high compared to the crude venom of the Colombian widow spider of the same genus. Considering these results, it is important to determine the concentration of the north American black widow sample to find the exact position of the bands in the electrophoresis. The band pattern observed is different between the two species between
17 and 70 kDa, this could indicate that the molecules responsible for the symptomatology described as latrodectism are located in the high molecular weight components, where the mayor toxins are located; because the symptoms that the bioassays showed in this study are consistent with the ones described for the syndrome. It is also interesting to note that the protein composition is not the same in the two species.
There are other 22 protein bands in the gel that need further studies to accomplish complete identification. There are many components that have a similar molecular weight and completely different structure and function in the venom of the spider (Haney et al.
2014). There are other compounds that have been described in other species of this genera
42
like adenosine, guanosine, inosine and 5-hidroxitriptamine (Rash and Hodgson 2002;
Bonnet 2004). The venom also contains a variety of enzymes like hyaluronidase that increases the diffusion of the injected material in the skin, gamma-aminobutyric acid that modifies and blocks the neuronal receptors in crustaceans (Bonnet 2004; Duan et al.
2006), and non-proteic components that are found in low concentrations; like serotonin that plays an important role in pain sensation and increases the defensive action of the toxin, this allows the penetration and distribution of the protein components within the bloodstream (Bonnet 2004). Also, the low molecular weight protein components, known as Latrodectins, are thought to enhance the toxicity, but they are not toxic alone (Haney et al. 2014).
According to the results of this study, it can be stated that the Colombian black widow studied in this investigation, could have medical importance because of the results obtained in the mice bioassays. In order to determine with certainty which toxins are present in the venom of this Latrodectus species, their concentrations and to find other components in the venom; the research studies should be continued.
Acknowledgments
All specimens were collected under collection permits emitted by the Colombian
Authority of Environmental Licenses ANLA.
Conflict of Interest
The authors declare no conflicts of interest.
43
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48
Chapter 1 Figure Legends
Figure1. Individual recipient with water receptacle.
Figure 2. Modified forceps for the venom extraction procedure.
Figure 3. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) with Coomasie Coloration; well 1 and 2: control sample from Bothrops asper, well 3:
5.075 µg of protein from Latrodectus sp. Venom, well 4: 3.045 µg of protein from
Latrodectus sp. Venom, well 5: 1.015 µg of protein from Latrodectus sp. Venom, well
6: 66 kDa weight marker, well 7: 170 kDa weight marker, well 8: empty, well 9 and 10: crude venom of L. mactans used as control. Blue rectangle showing bands at ~ 130 kDa, where α-latrotoxin should appear.
Figure 4. Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) with Silver Coloration; well 1 and 2: control sample from Bothrops asper, well 3: 5.075
µg of protein from Latrodectus sp. Venom, well 4: 3.045 µg of protein from
Latrodectus sp. Venom, well 5: 1.015 µg of protein from Latrodectus sp. Venom, well
6: 66 kDa weight marker, well 7: 170 kDa weight marker. Brown rectangle showing bands at ~ 130 kDa, where α-latrotoxin should appear.
49
Chapter 1 Table Legends
Table 1. Weight and age groups of mice for the toxicity tests in the bioassays.
Table 2. Symptoms presented by each group of experimental models after direct bite.
Means are rounded to nearest whole number.
Table 3. Identification of the molecular weight of the proteins present in the venom of
Latrodectus sp. from Colombia.
50
Chapter 1 Figures
Figure 1.
Figure 2.
Figure 3.
51
Figure 4.
52
Chapter 1 Tables
Table 1.
Group Weight (g) Age (days) 1 1.4-1.5 3-4 2 18-22 20-25 3 25-30 30-35
Table 2.
Group Symptom Mean Time (min) SD 1 Diaphoresis 3 0.35 Redness at the bite site 4 0.07 Salivation 4 0.21 Spasms 6 0.14 Erratic movements 7 0.57 Death 20 1.41 2 Pain at the bite site 0 0 Redness at the bite site 5 1.70 Diaphoresis 15 3.61 Salivation 15 0.28 Increased heart rate 15 1.56 Spasms 30 1.91 Erratic movement 55 6.79 Difficulty breathing 60 4.03 Death 105 21.99 3 Pain at the bite site 0 0 Redness at the bite site 6 0.28 Diaphoresis 37 5.80 Salivation 37 4.95 Increased heart rate 50 2.59 Spasms 64 9.19 Notorious Pain 74 4.88 Erratic movement 90 14.00 Difficulty breathing 120 20.58 Paralysis 180 28.00 Death 207 9.76
Table 3.
Distance migrated Y MW in Da MW in kDa 4.62 5.113 129668.35 129.67 5.793 5.037 108995.88 109.00 6.138 5.015 103568.24 103.57 7.31 4.940 87069.69 87.07 8.137 4.887 77035.50 77.04
53
8.827 4.842 69554.29 69.55 9.275 4.814 65090.48 65.09 9.758 4.782 60598.32 60.60 10.965 4.705 50681.62 50.68 12.034 4.636 43262.72 43.26 12.758 4.590 38865.17 38.87 13.792 4.523 33348.35 33.35 14.585 4.472 29654.08 29.65 15.723 4.399 25055.96 25.06 16.585 4.343 22053.85 22.05 17.309 4.297 19812.14 19.81 18.861 4.197 15744.82 15.74 19.378 4.164 14584.60 14.58 20.033 4.122 13236.65 13.24 21.55 4.024 10573.90 10.57 22.24 3.980 9547.02 9.55 23.55 3.896 7863.85 7.86
54
Chapter 2:
Two new species of Latrodectus (Aranea: Theridiidae) from the dry
forests in the Magdalena Valley, Colombia
ALEXANDRA RUEDA1*, ADOLFO AMÉZQUITA2, VALENTINA MUÑOZ-
CHARRY3, DANIELA LOZANO1, MARÍA ISABEL VELÁSQUEZ-VÉLEZ1,
EMILIO REALPE1.
Corresponding authors:
Alexandra Rueda ([email protected])
Running Title:
New Latrodectus from Colombia
Cited references: 33
Figures: 10
Scope: Latrodectus phylogeny
New Taxa: 2 new species
Submitted to Journal: Invertebrate Systematics
55
Two new species of Latrodectus (Araneae: Theridiidae) from the dry
forests in the Magdalena Valley, Colombia
ALEXANDRA RUEDA1*, ADOLFO AMÉZQUITA2, VALENTINA MUÑOZ-
CHARRY3, DANIELA LOZANO1, MARÍA ISABEL VELÁSQUEZ-VÉLEZ1,
EMILIO REALPE1.
1 Laboratory of Zoology and Aquatic Entomology, Department of Biological Sciences,
Universidad de los Andes, AA 4976. Bogotá, Colombia.
2 Department of Biological Sciences, Universidad de los Andes, AA 4976. Bogotá,
Colombia.
3Biomics Laboratory, Department of Biological Sciences, Universidad de los Andes, AA
4976. Bogotá, Colombia.
*Corresponding author: [email protected]
Abstract
Black widow spiders are known to wide audiences for their medical importance and to spider taxonomists for the striking variation in coloration and low differentiation in internal morphology. Only two species were previously described in Colombia:
Latrodectus curacaviensis and the widely distributed L. geometricus, from the Caribbean coasts and mid-Andean valleys, respectively. The use of molecular data, for the first time
56
on Colombian species, revealed two candidate species of Latrodectus, which we further studied and describe here. A molecular phylogenetic analysis was conducted with
Bayesian Inference using partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI), and the mitochondrial ribosomal RNA gene 16S (16SrRNA). Using the phylogenetically closest species, we tested for reciprocal monophyly, estimated genetic divergence, and identified fixed sites within each gene. All species were genetically compared with the closest phylogenetic relative to give enough statically validation to the new species hypothesis. Despite the limited value of morphological traits in Latrodectus taxonomy and species delimitation, we provide here a detailed morphological description of male and female sexual organs, as well as general body traits for the two candidate species. New population reports are made for L. geometricus and L. curacaviensis in 10 departments in the country. Also, a synonymy for two South-American species, described originally in Argentina, is determined.
Key words: Molecular phylogeny, Morphological description, Black widow.
Resumen
Las arañas viudas negras son ampliamente conocidas por su importancia médica y son de gran interés en taxonomía por su variación en coloración y baja diferenciación en morfología. Sólo dos especies han sido descritas previamente en Colombia: Latrodectus curacaviensis y L. geometricus, en la costa Caribe y los valles inter-Andinos, respectivamente. El análisis de datos moleculares, usados por primera vez en especies colombianas, reveló dos especies candidatas de Latrodectus que estudiamos más a fondo y describimos en este manuscrito. Se realizó una filogenia por inferencia Bayesiana usando secuencias parciales de los genes mitocondriales citocromo c oxidasa subunidad
57
I (COI) y el gen mitocondrial ribosomal 16SrRNA. Con los clados más cercanos se evaluó monofilia recíproca, se estimó la divergencia genética y se identificaron sitios fijos dentro de cada gen. A pesar del valor limitado de los caracteres morfológicos en la taxonomía del género y la delimitación de especies, se realiza una descripción morfológica detallada de los órganos sexuales en hembras y machos, así como de caracteres corporales generales para las dos pesecies candidatas. Se realizan nuevos reportes de poblaciones para L. geometricus y L. curacaviensis en 10 departamentos del país. Además, se determina la sinonimia de dos especies descritas originalmente en Argentina.
Palabras clave: Filogenia molecular, descripción morfológica, viudas negras.
Short summary
The first approach to black widow systematics knowledge in Colombia. Two new species of Latrodectus are described, and new localities are reported. Morphological description is accomplished. Genetic distances are calculated between related taxa. All our results support the status of new species of L. garbae, sp. nov. and L. hurtadoi, sp. nov., and the synonymy of L. diaguita and L. corallinus.
Introduction
The black-widow spider genus, Latrodectus Walckenaer, 1805 (Araneae:
Theridiidae), are widely known with regard to the powerful venom they produce. The genus contains 31 species described to date (Platnick 2017); the taxonomy remains complex, because much of the variation in coloration, abdomen color patterns, and the abundance of setae, appears to be continuous throughout wide geographical ranges (Levi
1959). Very early, 22 extant species had been collapsed into just six, and many of which were considered cosmopolitans (Levi 1959). Although taxonomists initially paid
58
particular attention to the female and male genitalia (epigynum and pedipalps), the high similarity in reproductive structures for most species undermined their value for species identification (Levi 1983). Molecular techniques have thus become a crucial tool for the determination of species boundaries and the recognition of new species in the genus (Garb et al. 2004).
Two species of Latrodectus are currently listed in Colombia, L. curacaviensis
(Müller, 1776) and L. geometricus Koch, 1841. There are also reports of Latrodectism syndrome, but the species involved is not reported (Quintana and Otero 2002; Marie and
Vetter 2015). During field expeditions in Colombia, we found populations of Latrodectus with a distinctive color pattern not resembling any of the Colombian species. Since coloration and reproductive structures are not sufficient traits for species description, we conducted molecular analysis on collected specimens that erected them as candidate species. The new morphotypes were found in dry to very dry forests: Tatacoa (Huila) and
Mesa de los Santos (Santander). At about 440 meters above sea level (masl), Tatacoa is characterized by two rainy seasons per year with annual rainfall between 1000–1500 mm
(average data from 1981-2010, IDEAM 2017). The Latrodectus spider found here had been reported in medical articles more than a century ago, under the common name of
“coya” (Aguirre-Plata 1914; Hamburger 1938). Its venom was described as strong and able to produce mortal spasms (Gilij 1955). The small rate of human accidents with this species is intriguing, even casting doubts on its existence (Boussingault 1985). At about
1480 masl, Mesa de los Santos receives less than 1000 mm of annual rainfall (IDEAM
2017); the intense solar radiation, strong winds, and high evapotranspiration favors arid conditions (Cuatrecasas 1958; Casas-Pinilla and Ríos-Málaver 2017). There are no previous reports of any accident related with venomous spiders, or any historical records of the colonization or establishment of a population of black widow spiders in this region.
59
Dry forests are an endangered ecosystem in Colombia, with less than 8% of the original habitat remaining (Pizano et al. 2014). In addition, little is known about the natural history, ecology, and biology of Latrodectus in the country. The lack of sound information on them, as well as the ecological and medical importance of the genus, offer enough justification to test the taxonomic identity of these morphotypes and to formally describe them. We further provide a phylogenetic framework for our discussion, including new population reports for currently recognized species and the new taxa.
Materials and Methods
Latrodectus spiders had been reported in dry forests of Colombia (Quintana and
Otero 2002; Rueda et al. 2017) including the arid environments in Tatacoa (Aguirre-Plata
1914; Hamburger 1938; Gilij 1955; Boussingault 1985). We conducted field work to sample the major dry forests of the country that were comparable with regard to climatic conditions (Pizano et al. 2014, see Table S1) to the previously known localities of
Latrodectus. All specimens were collected under collection permits emitted by the
Colombian Authority of Environmental Licenses ANLA.
Most webs were found within 50 cm above the ground and anchored on rocks or small plants (Rueda et al. 2017). Only L. geometricus was found near or within human habitats. Between 2010–2016, we collected specimens with forceps and placed them in a glass vial with absolute ethanol. They were then preserved at -20 C in the Zoology and
Aquatic Entomology Lab (LAZOEA) at the Universidad de los Andes (Bogota,
Colombia), until the morphological and molecular analyses were conducted. Preserved specimens were later deposited at the Natural History Museum ANDES, in the invertebrate collection (Universidad de los Andes), whereas DNA sequences were uploaded to GenBank (see Table S1 for accession codes). For several Latrodectus species,
60
we further received DNA aliquots from Jessica Garb (Garb et al. 2004) and whole specimens (L. hesperus Chamberlin & Ivie, 1935) from EVOLAB, University of
California, Berkeley, USA.
DNA extraction, PCR amplification and sequencing: The legs or cephalothorax of each specimen were used for DNA extraction with the Qiagen Kit
(DNeasy Blood & Tissue, Qiagen). The universal primers for COI: LCOI 1498: 5' GGT
CAA CAA ATC ATA AAG ATA TTG G 3' and LCOI 2198: 5' TAA ACT TCA GGG
TGA CCA AAA AAT CA 3', and for 16SrRNA: 16SF: 5' CGC CTG TTT AAC AAA
AAC AT 3' and 16SR: 5' CCG GTT TGA ACT CAG ATC ACG T 3' were used at 10
µM for the amplification of the mitochondrial genes. The PCR conditions used in an
INFINIGEN thermal cycler were as follows: initial denaturation at 94°C during 3 min,
35 cycles of 94°C during 45 s, 50°C during 50 s, and an extension of 72°C for 1 min, ending with a final extension of 72°C during 5 min for COI. Annealing temperature for
16S was of 47°C keeping the rest of the cycle. The PCR products were purified using the
EXO-SAP method and sent for sequencing to MACROGEN INC. (Seoul, Korea).
Phylogenetic reconstruction and genetic analysis: Latrodectus COI sequences were downloaded from NCBI and combined with our own sequences (Table S1). The whole set was cleaned and assembled using Geneious R 11 (Kearse et al. 2012), aligned using Clustal W (Thompson et al. 1994), and tested for model evolution with JModelTest
(Posada 2008); we used AICc to select the substitution model that better fitted the samples. A phylogenetic analysis with Bayesian Inference was conducted on BEAST
1.8.4 (Drummond and Rambaut 2007), using 50 millions of generations and sampling trees each 1000; burn-in was calculated using TRACER (Rambaut et al. 2014). Kimura
2-Parameters genetic distances were calculated among studied species with Mega 7
(Kumar et al. 2015). A threshold of 2% of genetic difference was used as molecular
61
evidence of separate lineages (Barrett and Hebert 2005). Median Joining Networks of assemblage for COI and 16SrRNA sequences were built using PoPArt software (Leigh and Bryant 2015). Also, genetic indexes such as Fst, Haplotypic diversity were calculated using DNAsp 5 (Librado and Rozas 2009).
Morphological analyses: The female epigyna were dissected and made transparent using a solution of 10% KOH, leaving them in simmer for five days, and then clearing in clove oil. Male palps were expanded through repeated immersions in the KOH solution and distilled water. We then noted character states for male pedipalp, external and internal female genitalia, and color pattern; the structures were photographed and measured in a Leica EZ4HD stereoscope.
Results
We found evidence to recognize four species of Latrodectus in Colombia, two of which are currently named L. curacaviensis, L. geometricus. They were found along the
Magdalena valley mostly in dry and warm places. The only species occurring in habitats with a wide range of temperatures was L. geometricus, from Silvania, Cundinamarca
(average temperature of 19°C ) (IDEAM 2017) to Tatacoa (average temperature of 37°C)
(IDEAM 2017). We found new populations for L. geometricus in 10 localities, and for L. curacaviensis 1 new locality (see Table S1 and Figure S4). The candidate species found in the Tatacoa desert was first found living in sympatry with L. geometricus, but after
2016 no specimens were found again.
Phylogenetic analysis: A total of 112 sequences of COI and 69 sequences of 16S were used for the independent and concatenated alignments used for the phylogenetic inference. All new sequences were made available on GenBank (sequence codes in Table
S1). The best substitution model according to AICc test for COI was TMV+I+G; for 16S
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was GTR+G. The phylogenetic hypothesis resulting from the concatenated alignment
(Figure S1) grouped together all Latrodectus sequences with a posterior probability (pp) of 1. The two major clades described in Garb et al. (2004) were recovered with a pp of 1: geometricus and mactans.
Beyond the currently named species, we recovered two monophyletic groups of
Colombian samples. One group (Morphotype 1 – MT1) occurred in and near Tatacoa
(Departaments of Huila and Tolima, central Andes of Colombia) in sympatry with L. geometricus; it appeared sister to the Argentinian species L. corallinus Abalos, 1980 and
L. diaguita Carcavallo, 1960, and the including taxon was in turn reciprocally monophyletic to L. curacaviensis (Figure 1). The second group of unnamed samples
(Morphotype 2 – MT2) was obtained from Mesa de los Santos (Barichara, Colombia), where it occurs roughly in the same area with L. curacaviensis and L. geometricus; it was sister to the two North American lineages currently recognized as L. herperus Chamberlin and Ivie, 1935 (Figure 2). Complete concatenated and gene phylogenies are available in supplementary material.
New populations of MT1 were detected north of the desert in locations such as
Natagaima, Castilla, Aipe and Saldaña, and one specimen was collected in La Dorada,
Caldas. MT2 was only found in “La Mesa de los Santos”, in the same ecosystem with L. curacaviensis and L. geometricus, but the webs were not found near each other. The spiders were found away from human roads or constructions and the webs were not easily visible although the size of the female spiders are bigger than other species (see Table S1 and Figure S4). Genetic distance between L. corallinus and L. diaguita was of 0.1% with a Fst index of 0, and between these two species and L. curacaviensis the distances were
5.1% and 5%, with Fst’s of 0.97 and 0.99 respectively. The relation between L. curacaviensis and MT1 showed a genetic distance of 4.4% and and Fst of 0.96. Between
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the Argentinian species and MT1 the minimum genetic distance was of 1.7% with an Fst value of 0.85 (Table S6). The haplotype network for COI (Figure S5) shows 16 fixed sited between L. curacaviensis and MT1, and 6 between the Argentinian species and MT1
Also, one haplotype between the two Argentinian species is shared. Alignments and fixed sites for COI Alignment can be seen in Table S2.
The haplotype network for the 16SrRNA analysis (Figure S6) show 7 fixed sites between L. curacaviensis and MT1, a genetic distance of 1.8%, and an Fst index of 1.
Two diagnostic characters were obtained between MT1 and the two Argentinian species.
The lower genetic distance recovered between the Argentinian species and L. curacaviensis was of 1.8% with an Fst index of 0.87. The lower genetic distance between
MT1 and the Argentinian species was of 0.5% with an Fst of 0.87 (Table S6). Shared haplotypes are found in the two Argentinian species. To see the alignment and fixed sites see Table S3 and S6.
The hesperus clade, shows L. hesperus grouped with MT2 in three well defined groups for all analysis, and reciprocal monophyly was recovered for all trees (Figure 2 and S1-S3). Using COI gene fragment, L. hesperus from Canada, separates from L. hesperus United States with a genetic distance of 7.5%. The population of L. hesperus
Canada forms a monophyletic group with MT2 showing a genetic distance of 2.7% and a pp of 1 for all phylogenetic hypothesis. Also, there are 14 fixed sited between these two species (Figure 3, S4, S7), and 30 between the two populations of L. hesperus. The K2P genetic distance recovered between the Canada population and the US population is of
7.5% with an Fst index of 0.94 (Table S6, Figure S7). 16SrRNA alignment between MT2 and related taxa showed a genetic distance of 1.6% was obtained and an Fst index of 0.96, haplotype network showed 7 fixed sites (Figure S8, Table S5).
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Summarizing, the K2P genetic distances between our two unnamed lineages and their closest relatives were between 1.7–4.4% (Figure 3 and Table S6). The genetic alignment for COI (Table S2) revealed between 6 and 16 fixed sites that separated the unnamed specimens of Tatacoa from the Argentinian species and L. curacaviensis, respectively.
Based on the clear reciprocal monophyly between each of our two groups of unnamed spiders and other taxa, the long geographic distance, and the moderate to large genetic distance separating them, we looked for additional morphological evidence to formally describe them as new taxa. The results are shown below.
Systematics
Latrodectus garbae, sp. nov.
Holotype: Locality: Tatacoa “desert”, Huila, Colombia, (502 m), (3°13’0.23”N,
75°8’0.72”W), nine females and eight males, collected by Alexandra Rueda, (ANDES-
IN 2990, 2987, 2988, 2989, 2991, 2992, 2993, 2994, 2995, 2998, 2999, 3000, 3001, 3002,
3003, 3004, 3005).
Paratypes: One female and one male from the same locality, Alexandra Rueda
(ICN- Ar 8124, 8125).
Etymology: The species is dedicated to Jessica Garb, the scientist who built the first molecular phylogeny of the genus and has greatly contributed to the knowledge of black widows systematics and evolution.
Diagnosis and definition: L. garbae genitalia is similar to other Latrodectus species specially to L. curacaviensis. The spermatic ducts of the female have three coils in L. garbae as well as a dorsally membranous shaped depression in the distal segment of
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the third coil (Figure 5A); also, the first spermatec is greater than the second. Regarding male genitalia, the embolus has three coils; the conductor has the distal part with a pronounced curvature to the prolateral direction (Figure 4D, Figure 5C). Latrodectus garbae’s color pattern consists of a black background in the ventral view where the red hourglass is seen (Figure 4C). The shape of the hourglass is not as marked as in other
Latrodectus species, but more rounded. On the dorsal side, the background of the abdomen is red and there are six easily recognizable spots forming a symmetrical arrangement in the antero-posterior axis (Figure 4A). The spots located near the pedicel are the largest ones and can get fused with the black background of the ventral portion
(Figure 4B) of the body, forming an arrow-like structure, which points to the anterior portion of the body; the smallest spots are in the posterior portion of the abdomen body.
Males show the same patterns in the abdomen, but the spots do not fuse. The color and the pattern in the abdomen are retained until maturity (Figure 6). In 10 egg sacs we inspected, the shape was spherical, with wooly appearance and white coloration, and the surface did not have ornamentation. Average diameter of 8.02 ± 0.16 mm.
Description: Female (N=20): Small size spiders, size inferred from the carapace width (Hagstrum 1971). Bright brown carapace with dark brown color in the thoracic furrow and radial furrow, equally long as wide (cephalothoracic coefficient T =1.04 ±
0.08, after Melic 2000) with oval form. Dark brown to black sternum. Sub globular abdomen with distinctive color pattern, totally covered with setae. Dark brown to black legs with joints of the same color. Epigynum (N=10) heavily sclerotized epigynal plate with sub oval form, wider than long, covered with thin setae, oval opening of the atrium with sclerotized margins. Spermatheca in V structure, close together in the base but not in contact. Copulatory ducts have three coils, the third coil is shorter that the other two, when the duct is expanded, the distal portion is observed to have a circular flattened form,
66
membranous like, and dorsally projected. One out of 10 examined females presented just two coils in the spermatic ducts. Carapace length (N=20 females): 2.43 ± 0.31 (mean ± sd) mm (range 1.85 – 3.17 mm); width: 2.33 ± 0.20 mm (range 2.03 – 2.73 mm). Carapace coefficient: 1.04 ± 0.08 (range 0.81 – 1.16). Femur I length: 4.55 ± 0.46 mm (range 3.75
– 5.39 mm). Patella I length: 1.16 ± 0.15 mm (range 0.91 – 1.47 mm). Tibia I length: 3.79
± 0.40 mm (range 3.30 – 4.84). Sternum length: 1.51 ± 0.15 mm (range 1.29 – 1.92 mm); width: 1.28 ± 0.14 mm (range 1.10 – 1.54 mm). Male (N=10): Male size much smaller than females. Dark brown carapace equally long as wide (cephalothoracic coefficient mean T: 0.96 ± 0.01). Subglobular Abdomen with color pattern equal to that of females.
Dark brown legs in proportion to the body much longer than females. Pedipalp similar to
L. curacaviensis. The embolus of the male palpus have three coils, same as copulatory ducts in females (Bhatnagar and Rempel 1962). Total length: 2.74 ± 0.19 mm (range 2.32
– 3.22 mm). Carapace length: 1.02 ± 0.12 (range 0.92 – 1.27 mm); width: 1.07 ± 0.13
(range 0.94 – 1.32 mm). Carapace coefficient: 0.95 ± 0.06 (range 0.73 – 1.04). Tibia I length: 2.25 ± 0.3 mm (range 1.0 – 2.87). Patella I length: 0.58 ± 0.11 mm (range 0.46 –
0.67 mm). Patella- Tibia index: 2.77 ± 0.90 (range 1.55 – 3.31).
Distribution: Interandean Magadalena valley of southwestern Colombia, in
Departmentos of Huila, Tolima and Caldas. Found in hot and dry places, away from human structures and near to the ground in small vegetation or rocks.
Natural history: Latrodectus garbae, sp. nov. was first found in 2009 living in sympatry with L. geometricus in Tatacoa. After 2016, no specimens were found again, but new populations were detected north to the type locality, in Natagaima, Castilla, Aipe and Saldaña; and one specimen was collected in La Dorada, Caldas.
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Latrodectus hurtadoi, sp. nov.
Holotype: Female from Mesa de los Santos, Santander, Colombia, (1400 m),
(6°47' 54.6714”N, 73°9'46.6554”W), collected by Alexandra Rueda (Collection Number
AR_1).
Paratypes: Eight females and one male collected by Alexandra Rueda (Collection
Number AR_2-10).
Etymology: The name of the species is a dedication to Andrés Hurtado García,
Colombian journalist, ecologist, alpinist, and nature photographer. In honor to his great labor in education and protecting the environment, and because of his passion for life and spiders.
Diagnosis and definition: Genitalia are similar to other Latrodectus species. In all males dissected, the embolus was recovered inside the epigynum. Male embolus has three coils, showing a loop in the end of the third coil (Figure 7A and 7B). The conductor and the terminal apophysis are close together, and both show pronounced curvature to the prolateral direction (Figure 7A and 7B). The color pattern is very similar to L. hesperus, even in the ontogenetic change. Spiderlings show a light brownish coloration, similar to
L. geometricus, but the color pattern in the abdomen show lateral orange, red or white stripes surrounded by a darker border. The first stripe, near the pedicel goes around the abdomen and ends up in the ventral portion. Second and third stripe follow the lateral segment of the body, and the third and bigger goes from the spinnerets to the middle of the dorsal portion in the abdomen, following the middle longitudinal axis of the body.
Carapace light brown with a darker border and a longitudinal darker line going through the carapace furrow. Legs light brown with darker portions in joints and in the middle of each segment (Figures 7 - 9). After a couple of molts, the abdominal background color turns darker and the lateral stripes start to fade. The longitudinal stripe turns reddish
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(Figure 7). When maturity is reached the longitudinal red stripe is retained; also, the transversal stripe near the pedicel, can or cannot be present in an adult female (Figure 9).
The legs and cephalothorax turn dark brown to black. The shape of the hourglass is marked and noticeable (Figure 9). Males keep the spiderling coloration until maturity
(Figure 10). Egg sac: (N=6) Spherical white egg sac with wooly organized appearance, the surface does not have ornamentation. Average diameter of 7.56 ± 0.98mm.
Description: Female (N=20): Big size spiders. Bright black to dark brown carapace, with dark black color in the thoracic furrow and radial furrow, longer than wider
(cephalothoracic coefficient T =1.12 ± 0.09) with oval form. Black sternum longer than wide. Sub globular abdomen with distinctive color pattern, totally covered with setae.
Dark legs with joints of the same color or with darker joints and darker marks in each segment. Ontogenetic color change in the abdomen: juvenile spiders show a light brown coloration, similar to L. geometricus in cephalothorax and legs, but the abdominal color pattern is different. Females change the background color to darker color with each molt, and the dorsal pattern starts to have yellow line markings on the sides of the abdomen and a red line in the middle following the longitudinal axis. When they are reaching sexual maturity the color pattern is lost, leaving only the red line across the abdomen and the transversal line near the pedicel. The hourglass is big and defined on a black background.
The epigynum (N=10) is heavily sclerotized, epigynal plate with sub oval form, wider than long covered with thin setae, oval opening of the atrium with sclerotized margins.
Spermathec in V structure, close together in the base but not in contact. Copulatory ducts have three coils, the third coil is located in the back of the spermatec and is shorter that the other two; when the duct is expanded, the distal portion is observed to have a circular flattened form, membranous like, and dorsally projected. All of the dissected epigyna had the male coils inserted (males loose structure during copula). Carapace length: 2.90 ±
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0.43 mm (range 1.63 – 3.64 mm); width: 2.61 ± 0.37 mm (range 1.37 – 3.02 mm).
Carapace coefficient: 1.12 ± 0.09 (range 0.91 – 1.26). Femur I length: 5.00 ± 0.48 mm
(range 3.80 – 5.88 mm). Patella I length: 1.50 ± 0.17 mm (range 0.96 – 1.77 mm). Tibia
I length: 4.17 ± 0.41 mm (range 3.38 – 4.97). Sternum length: 1.94 ± 0.32 mm (range
1.40 – 2.60 mm); width: 1.54 ± 0.31 mm (range 1.23 – 2.60 mm). Male: Just one male reared in captivity. Male size much smaller than females (3,56 mm). Light brown carapace longer than wider (cephalothoracic coefficient T: 1.26). Subglobular abdomen with coloration similar to spiderlings. Light brown legs in proportion to the body much longer than females. Males keep the light brown coloration all their life. The embolus of the male palpus had three coils same as copulatory ducts in females. Total length: 3.56 mm. Carapace length: 1.29 mm; width: 0.78 mm. Carapace coefficient: 1.21. Tibia I length: 2.53 mm. Patella I length: 0.71 mm. Patella-Tibia index: 2.08.
Distribution: Northern Colombia, Department of Santander, Mesa de los Santos, located in hot, dry places away from human structures and near to the ground in small rocks; the habitat lacked tree or shrub vegetation and was mostly sandy.
Natural history: L. hurtadoi, sp. nov. was only found in “La Mesa de los Santos”, in the same ecosystem with L. curacaviensis and L. geometricus, but the webs were not found near each other. The spiders were always away from human roads or constructions, in a field full of little stones on the ground. The webs were not big, and spiders were not easily visible although female size was bigger than in the other species. All the localities and sampling sites for each species are found in Table S1.
Discussion
The taxonomy of Latrodectus has been difficult given the highly conserved morphology among the species (Levi 1983), as well as the high intraspecific variation of
70
coloration and genitalia: across-species comparisons have failed to find a character that works for the differentiation of species (Gerschman and Schiapelli 1965). Our description of male and female genitalia of L. garbae, sp. nov. and L. hurtadoi, sp. nov., revealed many similarities to other Latrodectus, even though, the pedipalp and epigynal structures can be sufficiently distinguishable from other Latrodectus spiders.
Molecular techniques support the description of new species, especially when species have multiple morphotypes, as in Latrodectus (Melic 2000). COI and 16SrRNA genes allow the discrimination and differentiation of black widow spiders (Croucher et al. 2004; Barrett and Hebert 2005; Padial et al. 2010). The COI marker has a good phylogenetic signal, but for intraspecific analysis does not have any resolution; moreover, the use of just one marker could bias phylogenetic analyses (Simon et al. 2006; Hajibabaei et al. 2007). The trees presented have a similar topology compared to Garb et al. (2004): two main clades are recovered in all trees, geometricus and mactans (Complete phylogeny in Figure S1).
The geometricus clade groups all L. geometricus, regardless of the collecting site, and the sister clade L. rhodesiensis Mackay, 1972. All sequences of L. geometricus from
South America, are located in the same clade with a pp of 0.85. The origin of this species is yet unknown, and its wide distribution and invasion history makes it difficult to determine (Marie and Vetter 2015). All the Colombian L. geometricus were grouped in one clade with a pp of 0.85. New population reports were made for the country in Meta,
Cundinamarca, Vichada, Guajira, Bolivar, and Santander. Also, a new report for L. curacaviensis in the Department of Santander. For specific localities see Table S1 and
Figure S4.
In the mactans clade, L. mactans groups with L. hesperus clade with a pp of 0.44.
The relation between L. bishopi and L. variolus is recovered in both trees with a pp of
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0.88 in the concatenated alignment and of 0.92 in the COI alignment. The next clade we can see in Figure S1 is the hesperus clade, that includes L. hesperus and L. hurtadoi, sp. nov. showing three distinctive groups, L. hesperus USA, L. hesperus Canada, and L. hurtadoi, sp. nov. The genetic distance between L. hesperus from USA and Canada is of
8%, and it has been suggested in previous studies that they belong to different species
(Barrett and Hebert 2005). Also, there are 32 fixed sites between the sequences of these two species in the COI gene fragment. These results support the hypothesis that they belong to different species, but specimen revision is needed. A deeper analysis is needed in order to determine the correct identity of these species.
L. hurtadoi, sp. nov. show a genetic distance of 7% with L. hesperus USA and of
3% with L. hesperus Canada. Also, the percentage of divergence for COI is of 5.79% and
2.51%. There are 30 and 13 fixed sites between these species for COI and 16S respectively (Table S6). These analysis, with the reciprocal monophyly recovered for all trees, the morphological analysis, and the geographical location, supports the hypothesis of the new species status for L. hurtadoi, sp. nov.
The South American clade shows that L. mirabilis, L. variegatus and L. thoracicus group together with a pp=1 for all trees. This clade should be studied deeper, results show that L. thoracicus and L. variegatus may belong to the same species; type specimens should be checked, and deeper genetic analyses made. The position of L. antheratus
(Badcock, 1932) changes between concatenated, COI and 16SrRNA inference trees. In the concatenated and COI analyses it forms a monophyletic clade with the Colombian species (pp=0.94, pp=0.87 respectively), but in the 16SrRNA inference tree the topology supports its position next to L. mirabilis and L. variegatus with a pp=0.64. The curacaviensis clade groups L. curacaviensis¸ L. corallinus¸ L. diaguita and L. garbae sp. nov. The internal topology of this group was recovered as reciprocal monophyly in all
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trees with the lowest pp of 0.99. L. curacaviensis appears as sister of the other species
(pp=1 in all topologies). L. corallinus and L. diaguita appear together with internal topologies not defined. The genetic distance between these two Argentinian species is 0% and they show a value of Fst of 0 and a 0% in genetic divergence; also, there are no fixed sited between these two species. Even though, the specimens collected in (Garb et al.
2004) showed a difference in the egg sac morphology, as described in (Abalos 1980), where L. corallinus shows a “spiked” egg case, while L. diaguita has a smooth egg case; there are enough reasons to synonymize these two species as L. diaguita. L. garbae sp. nov. shows a genetic distance of minimum 2% and a minimum Fst value of 0.86 with very low gene flow and at least 5 fixed sites for COI and 2 for 16S. Reciprocal monophyly was recovered for all trees and geographical location changes. These reasons support the hypothesis of the new species status for L. garbae sp. nov. Altogether, our molecular data and phylogenetic hypothesis contribute to the knowledge and increase the species sampling for COI and 16S markers in the genus.
Acknowledgments
We thank the Biological Science Department for funding part of this investigation and field trips. We received help from Oscar Ramos, Diego Gomez, Yiselle Cano, Nestor
Galindo and Diana Stasiukynas, finding the spiders and in the collecting process. We thank Oscar Ramos for his great collaboration and information regarding sampling sites.
Also, Diego Parra for being the collector of the first specimens of L. garbae, sp. nov. in the Tatacoa desert for this study.
Conflict of Interest
The authors declare no conflicts of interest.
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Chapter 2 Figure legends
Figure 1. Bayesian Inference fragment of the Bayesian phylogenetic tree for a group of
Latrodectus spiders, using the concatenated alignment of COI and 16S fragment as markers. The fragment shows the Latin-American clade with L. curacaviensis and one of the Colombian candidate species. Node numbers denote posterior probabilities. The complete phylogeny is shown in Figure S1.
Figure 2. Bayesian Inference fragment of the Bayesian phylogenetic tree for a second group of Latrodectus spiders, using the concatenated alignment of COI and 16S fragment as markers. The fragment shows the North American hesperus clade with L. hesperus and the second of the Colombian candidate species. Node numbers denote posterior probabilities. The complete phylogeny is shown in Figure S1.
Figure 3. The type locality of the two candidate species of Latrodectus at Mesa de los
Santos (black dot) and Tatacoa (yellow dot), both in dry forests of Colombia. Distribution and K2P genetic distances to the phylogenetically closest species is also shown.
Figure 4. Latrodectus garbae, sp. nov. A: Abdominal pattern in dorsal view. B:
Abdominal pattern in lateral view. C: Abdominal pattern in ventral view. D: Pedipalp inretrolateral view. Pictures by Alexandra Rueda.
Figure 5. Latrodectus garbae, sp. nov. reproductive characters description: A: Epigynum dorsal view with path scheme of spermatic ducts. Scale bar: 0.2 mm B: Epigynum ventral view. Scale bar: 0.2 mm C: Right palp prolateral view. Embolus (em), conductor (cd),
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terminal apophysis (t. ap), cybium (cm). Scale bar: 0.2 mm. Drawings by Valentina
Muñoz.
Figure 6. Latrodectus garbae, sp. nov. Male abdominal pattern in dorsal view. Picture
Alexandra Rueda.
Figure 7. Latrodectus hurtadoi, sp. nov. reproductive characters description: A: Right palp in frontal view, B: dorsal view. Embolus (em), conductor (cd), terminal apophysis
(t. ap), cybium (cm). C: Epigynum dorsal view of spermatic ducts. Drawings by
Valentina Muñoz.
Figure 8. L. hurtadoi, sp. nov. Left: Spiderling with light brownish coloration. Right:
Juvenile showing a darker abdominal background, and cephalothorax remains light brownish. Pictures by Alexandra Rueda.
Figure 9. L. hurtadoi, sp. nov. Female adult. Left: Spinneret view, two little red stripes surrounding the main longitudinal mark. Hourglass. Right: Lateral view of the abdomen, specimen retained the transversal white stripe until maturity. Pictures by Alexandra
Rueda.
Figure 10. L. hurtadoi, sp. nov. Adult male reared in laboratory. Left: Spiderling coloration retained until maturity. No ontogenetic color change in males. Right: Male pedipalp. Pictures by Alexandra Rueda.
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Chapter 2 Figures
Figure 1.
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Figure 2.
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Figure 3.
Figure 4.
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Figure 5.
Figure 6.
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Figure 7.
Figure 8.
Figure 9.
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Figure 10.
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Chapter 2 Supplementary Material Files:
Two new species of Latrodectus (Aranea: Theridiidae) from the dry
forests in the Magdalena Valley, Colombia
Authors: Alexandra Rueda1*, Adolfo Amézquita2, Valentina Muñoz-Charry3, Daniela
Lozano1, María Isabel Velásquez-Vélez1, Emilio Realpe1.
1 Laboratory of Zoology and Aquatic Entomology, Department of Biological Sciences,
Universidad de los Andes, AA 4976. Bogotá, Colombia.
2 Department of Biological Sciences, Universidad de los Andes, AA 4976. Bogotá,
Colombia.
3Biomics Laboratory, Department of Biological Sciences, Universidad de los Andes, AA
4976. Bogotá, Colombia.
*Corresponding author: [email protected]
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Table S1. Species and sequences used in this study, species ID, Collection locality, and NCBI Accession numbers for COI and 16S sequences. Voucher number is given for the species that were sequenced by us.
GenBank GenBank Species Collecting site Author Voucher Accession # COI Accession # 16S
Crustulina sticta Yonkers, USA Arnedo et al. 2004 - - AY230947 Santiago del Estero, Argentina This study #009 COI 16S L. antheratus Santiago del Estero, Argentina This study #007 COI 16S
L. bishopi Florida, USA Garb et al. 2004 - AY383060 - Cruz del Eje, Argentina This study #20 COI 16S L. corallinus Santiago del Estero, Argentina This study #13 COI 16S Mesa de los Santos, Santander, Colombia This study 108 - 16S
Mesa de los Santos, Santander, Colombia This study 103 - 16S
Barichara, Santander, Colombia This study 56 COI - Mesa de los Santos, Santander, Colombia This study 111 - 16S
Barichara, Santander, Colombia This study 55 COI -
Mesa de los Santos, Santander, Colombia This study 113 - 16S L. curacaviensis Barichara, Santander, Colombia This study 57 COI -
Mesa de los Santos, Santander, Colombia This study 110 - 16S
Barichara, Santander, Colombia This study 61 COI - Mesa de los Santos, Santander, Colombia This study 67 COI -
Barichara, Santander, Colombia This study 54 COI -
Barichara, Santander, Colombia This study 60 COI - Barichara, Santander, Colombia This study 63 COI -
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Barichara, Santander, Colombia This study 65 COI - Barichara, Santander, Colombia This study 58 COI -
Barichara, Santander, Colombia This study 64 COI -
Cartamarca, Argentina This study #002 COI 16S L. diaguita Cartamarca, Argentina This study #003 COI 16S
Tatacoa desert, Huila, Colombia This study 28 COI -
Tatacoa desert, Huila, Colombia This study 27 COI - Tatacoa desert, Huila, Colombia This study 33 COI -
Tatacoa desert, Huila, Colombia This study 29 COI -
Tatacoa desert, Huila, Colombia This study 35 COI - Tatacoa desert, Huila, Colombia This study 39 COI -
Tatacoa desert, Huila, Colombia This study 38 COI -
L. garbae sp. nov. La dorada, Caldas, Colombia This study 2 - 16S Tatacoa desert, Huila, Colombia This study 93 - 16S
Tatacoa desert, Huila, Colombia This study 24 - 16S
Tatacoa desert, Huila, Colombia This study 23 - 16S Tatacoa desert, Huila, Colombia This study 19 - 16S
Tatacoa desert, Huila, Colombia This study 22 - 16S
Tatacoa desert, Huila, Colombia This study 26 COI 16S Tatacoa desert, Huila, Colombia This study 31 COI 16S
Natagaima, Tolima, Colombia This study 141 COI 16S
L. geometricus Honolulu, Hawaii, USA Garb et al. 2004 - AY383046 -
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Georgia, USA Blackledge et al. 2009 - FJ607567 FJ607456 Santiago del Estero, Argentina This study #016 COI 16S
Roodeport, South Africa This study #097 COI 16S
Roodeport, South Africa This study #099 COI 16S Florida, USA This study #034 COI 16S
Florida, USA This study #033 COI 16S
San Diego, CA, USA Garb, Hayashi 2013 - KC414076 - Puerto Gaitán, Meta, Colombia This study 130 COI -
Girardot, Cundinamarca, Colombia This study 117 COI 16S
Tatacoa desert, Huila, Colombia This study 16 COI 16S Aipe, Huila, Colombia This study 132 - 16S
Girardot, Cundinamarca, Colombia This study 118 COI -
Puerto Carreño, Vichada, Colombia This study 127 COI 16S Riohacha, Guajira, Colombia This study 115 COI 16S
Puerto Carreño, Vichada, Colombia This study 128 COI -
Cartagena, Bolivar, Colombia This study 121 COI - Girardot, Cundinamarca, Colombia This study 119 COI 16S
Cartagena, Bolivar, Colombia This study 122 COI 16S
Aipe, Huila, Colombia This study 133 - 16S Aipe, Huila, Colombia This study 135 - 16S
Tatacoa desert, Huila, Colombia This study 30 COI 16S
Tatacoa desert, Huila, Colombia This study 32 COI 16S
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Girardot, Cundinamarca, Colombia This study 14 - 16S Riohacha, Guajira, Colombia This study 116 COI 16S
Puerto Carreño, Vichada, Colombia This study 129 COI -
Barichara, Santander, Colombia This study 59 COI - Puerto Carreño, Vichada, Colombia This study 125 COI 16S
Puerto Lopez, Meta, Colombia This study 3 - 16S
Girardot, Cundinamarca, Colombia This study 15 - 16S Tatacoa desert, Huila, Colombia This study 36 COI -
Tatacoa desert, Huila, Colombia This study 4 - 16S
Tatacoa desert, Huila, Colombia This study 37 COI - Girardot, Cundinamarca, Colombia This study 120 COI 16S
Riohacha, Guajira, Colombia This study 114 COI -
Tatacoa desert, Huila, Colombia This study 34 COI 16S Puerto Carreño, Vichada, Colombia This study 126 COI 16S
New Zealand Vink et al. 2008 - EF121034 -
L. hasseltii New Zealand Vink et al. 2008 - EF121033 - Perth, Australia Garb, Hayashi 2013 - KC414078 -
California, USA Garb et al. 2004 - AY383070 -
California, USA Wheeler et al. 2017 - KY017971 - L. hesperus Riverside, CA, USA Garb, Hayashi 2013 - KC414080 -
New Mexico, USA Garb et al. 2004 - AY383071 -
Yarnell, AZ, USA Garb, Hayashi 2013 - KC414081 -
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California, USA This study 176 COI 16S California, USA This study 177 COI 16S
California, USA This study 175 COI 16S
California, USA This study 174 - 16S California, USA This study 178 COI 16S
British Columbia, Canada Blagoev et al. 2016 - HQ977092 -
British Columbia, Canada Blagoev et al. 2016 - HQ977091 - British Columbia, Canada Blagoev et al. 2016 - KP652641 -
British Columbia, Canada Blagoev et al. 2016 - KP656931 -
British Columbia, Canada Blagoev et al. 2016 - KP656707 - British Columbia, Canada Blagoev et al. 2016 - KP649032 -
British Columbia, Canada Blagoev et al. 2016 - KP646487 -
British Columbia, Canada Blagoev et al. 2016 - HQ977093 - Mesa de los Santos, Santander, Colombia This study 75 - 16S
Mesa de los Santos, Santander, Colombia This study 105 - -
Mesa de los Santos, Santander, Colombia This study 104 COI 16S Mesa de los Santos, Santander, Colombia This study 107 COI 16S
L. hurtadoi sp. nov. Mesa de los Santos, Santander, Colombia This study 72 - 16S
Mesa de los Santos, Santander, Colombia This study 71 - 16S Mesa de los Santos, Santander, Colombia This study 109 COI 16S
Mesa de los Santos, Santander, Colombia This study 106 COI 16S
Mesa de los Santos, Santander, Colombia This study 101 COI 16S
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Mesa de los Santos, Santander, Colombia This study 76 COI 16S Mesa de los Santos, Santander, Colombia This study 73 - 16S
Mesa de los Santos, Santander, Colombia This study 74 - 16S
Mesa de los Santos, Santander, Colombia This study 102 COI 16S Mesa de los Santos, Santander, Colombia This study 86 COI 16S
Mesa de los Santos, Santander, Colombia This study 112 COI 16S
Mesa de los Santos, Santander, Colombia This study 69 COI - New Zealand Vink et al. 2008 - EF121007 -
L. katipo New Zealand Garb et al. 2004 - AY383053 -
New Zealand Vink et al. 2008 - EF121009 - Mississippi, USA Garb et al. 2004 - AY383072 - L. mactans North Carolina, USA Garb et al. 2004 - AY383054 -
L. menavodi Madagascar, Comoro Island Garb et al. 2004 - AY383075 - L. mirabilis Buenos Aires, Argentina This study #15 COI 16S
Israel Garb, Hayashi 2013 - KC414082 -
L. pallidus Israel Garb et al. 2004 - AY383055 - Israel Garb et al. 2004 - AY383056 -
L. revivensis Israel Garb et al. 2004 - AY383078 -
L. rhodesiensis Namibia, South Africa Garb et al. 2004 - AY383079 - Chile Aguilera et al. 2009 - GU112098 -
L. thoracicus Chile Aguilera et al. 2009 - GU112099 -
Chile Aguilera et al. 2009 - GU112103 -
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Sayeret Shaaed, Israel Garb et al. 2004 - AY383081 - L. tredecimguttatus Zaragoza, Spain Garb et al. 2004 - AY383080 -
Bariloche, Argentina This study #005 COI 16S L. variegatus Santa Cruz, Argentina This study #004 COI 16S South Carolina, USA Garb et al. 2004 - AY383059 -
L. variolus Maryland, USA Garb et al. 2004 - AY383082 -
Ontario, Canada Blagoev et al. 2016 - KP651212 - Steatoda bipunctata Yorkshire, UK Arnedo et al. 2004 - - AY230951
Steatoda borealis North Carolina, USA Garb et al. 2004 - AY383085 -
Steatoda grossa Molokai, Hawaii, USA Garb et al. 2004 - AY383086 - Theridion longipedatum Iguaque, Colombia Arnedo et al. 2004 - AY231062 -
# DNA aliquots donated by Jessica Garb. Voucher number corresponds to Garb et al. 2004. ** Specimens donated by EVOLab, University of California, Berkeley, USA. * Specimens collected in this study.
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Figure S1. Bayesian Inference tree using the concatenated alignment of COI and 16S fragment as markers, analysis was run to 50 million of generations, each 1000 trees one was sampled. Tree without posterior burn-in. * show sequences from Colombian specimens and donated by EVOLab, University of California, Berkeley; # belong to DNA aliquots sent from Jessica Garb.
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Figure S2. Bayesian Inference tree using COI fragment as marker, analysis was run to 50 million of generations, each 1000 trees one was sampled. Tree without posterior burn-in. * show sequences from Colombian specimens and donated by EVOLab, University of California, Berkeley; # belong to DNA aliquots sent from Jessica Garb.
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Figure S3. Bayesian Inference tree using 16S fragment as marker, analysis was run to 50 million of generations, each 1000 trees one was sampled. Tree without posterior burn-in. * show sequences from Colombian specimens and donated by EVOLab, University of California, Berkeley; # belong to DNA aliquots sent from Jessica Garb.
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Table S2. Alignment of the partial sequence of cytochrome oxidase subunit I. Species included: L. corallinus, L. diaguita, L. curacaviensis and L. garbae sp. nov. Fixed sites are visible. # DNA aliquots donated by Jessica Garb. Voucher numbers correspond to Garb et al. 2004. *Specimens collected in this study.
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Table S3. Alignment of the partial sequence of 16S. Species included: L. corallinus, L. diaguita, L. curacaviensis and L. garbae sp. nov. Fixed sites are visible. # DNA aliquots donated by Jessica Garb. Voucher numbers correspond to Garb et al. 2004. *Specimens collected in this study.
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Table S4. Alignment of the partial sequence of COI. Species included: L. hesperus and L. hurtadoi sp. nov. Fixed sites are visible. Voucher numbers correspond to Garb et al. 2004. *Specimens collected in this study. *__CA,, Specimens donated by EVOLab, University of California, Berkeley. DNA sequences downloaded from GenBank show the Accession Number and sample locality of the specimen.
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Table S5. Alignment of the partial sequence of 16S. Species included: L. hesperus and L. hurtadoi sp. nov. Fixed sites are visible. *Specimens collected in this study. *-CA, Specimens donated by EVOLab, University of California, Berkeley. DNA sequences downloaded from GenBank show the Accession Number and sample locality of the specimen.
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Table S6. Table of genetic information and comparison between the two colombian new species and related taxa. Table includes K2P Genetic distance, posterior probability of the clades recovered for the tree topologies for each gene and the concatenated analysis, the percentage of genetic divergence and the number of fixed sites in the alignments shown above.
K2P Fst pp % of # of fixed Gene Species 1 Species 2 Genetic Population Concanetated/COI/16S divergence sites Distance Structure
L. corallinus L. diaguita 0 0.00 0.33/0.27/0.31 0% 0
L. corallinus L. garbae sp. nov. 0.02 0.86 1/1/1. 1.49% 6
L. corallinus L. curacaviensis 0.05 0.97 1/1/1. 4.71% 19 COI L. diaguita L. garbae sp. nov. 0.02 0.92 1/1/1. 1.24% 5
L. diaguita L. curacaviensis 0.05 1.00 1/1/1. 4.71% 19
L. garbae sp. nov. L. curacaviensis 0.04 0.96 1/1/1. 3.97% 16
L. corallinus L. diaguita 0 0.00 0.33/0.27/0.31 0% 0
L. corallinus L. garbae sp. nov. 0.01 0.67 1/1/1. 0.50% 2
L. diaguita L. garbae sp. nov. 0.01 1.00 1/1/1. 0.50% 2 16S L. corallinus L. curacaviensis 0.02 0.87 1/1/1. 1.75% 7
L. diaguita L. curacaviensis 0.02 1.00 1/1/1. 1.75% 7 L. garbae sp. nov. L. curacaviensis 0.02 1.00 1/1/1. 1.75% 7
L. hesperus USA L. hurtadoi sp. nov. 0.07 0.94 1/1/1. 5.79% 30
COI L. hesperus Canada L. hurtadoi sp. nov. 0.03 0.98 1/1/1. 2.51% 13 L. hesperus Canada L. hesperus USA 0.08 0.95 1/1/1. 6.18% 32
16S L. hesperus USA L. hurtadoi sp. nov. 0.02 0.97 1/1. 1.56% 6
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Figure S4. Location of the type locality of the two candidate species of Latrodectus sp., at the Mesa de los Santos in the northwest (red dot) and the Tatacoa desert in the southeast, and two more populations found (purple dots). New population reports for L. geometricus (green dots) and L. curacaviensis (orange dots).
Figure S5. Haplotype network of the partial sequence of COI for L. garbae sp. nov., and related taxa. We find two colors for L. garbae sp. nov.; green corresponds to the population from the Tatacoa desert, and blue to the population from Natagaima. 108
Figure S6. Haplotype network of the partial sequence of 16S for L. garbae sp. nov., and related taxa.
Figure S7. Haplotype network of the partial sequence of COI for L. hurtadoi sp. nov., and related taxa.
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Figure S8. Haplotype network of the partial sequence of COI for L. hurtadoi sp. nov., and related taxa.
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Chapter 3:
Niche overlap in two sympatric species of black widows in Colombia
using ecological and genomic data
ALEXANDRA RUEDA 1,3*, ADOLFO AMÉZQUITA2, HENRIK
KREHENWINKEL3, SUSAN KENNEDY3, ROSEMARY GILLESPIE3, EMILIO
REALPE1
Corresponding authors:
Alexandra Rueda ([email protected])
Running Title:
Latrodectus niche determination
Cited references: 57
Figures: 8
Scope: Latrodectus ecology
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Niche overlap in two sympatric species of black widows in Colombia
using ecological and genomic data
ALEXANDRA RUEDA 1,3*, ADOLFO AMÉZQUITA2, HENRIK
KREHENWINKEL3, SUSAN KENNEDY3, ROSEMARY GILLESPIE3, EMILIO
REALPE1
1 Laboratory of Zoology and Aquatic Entomology, Department of Biological Sciences,
Universidad de los Andes, AA 4976. Bogotá, Colombia.
2 Ecophysiology, behavior and herpetology Group, Department of Biological Sciences,
Universidad de los Andes, AA 4976. Bogotá, Colombia.
3 Environmental Sciences Policy and Management, University of California Berkeley
Mulford Hall, Berkeley, California, USA.
*Corresponding author: [email protected]
Abstract
The coexistence of very similar species challenges the fundamental prediction of competitive exclusion in ecology. When one of the species is invasive, the system offers an interesting opportunity to understand the ecological mechanisms underlying the population dynamics of invasive species. Two species of black widow spiders co-existed for more than 175 years in a single locality, one is considered invasive, and the other one disappeared in very recent years. We used ecological variables and molecular gut-content analysis to study niche partitioning between these two sympatric species. We estimated 112
diet by direct observation of prey remnants and, for the first time in this genus, by using molecular gut content metagenomics. We found significant environmental niche overlap between the two species in both microhabitat parameters and diet. Using metagenomics on spiders’ gut contents, we recovered a mean of 19.28% of prey reads in 33 specimens of Latrodectus sp. and 19 specimens of L. geometricus. Our results show that prey recovery for Latrodectus sp. was 15.55% of the total prey reads recovered, and the remaining 84.45% belong to L. geometricus specimens. Despite the difference in recovered prey reads, the prey community alpha diversities are very similar for both species, even at low taxonomic ranks. We expected differences in niche composition between the two sympatric species, but results support a complete overlap.
Key words: Competition, segregation, NGS, Latrodectus.
Resumen
La coexistencia de especies muy similares entra en conflicto con la predicción fundamental en ecología de la exclusión competitiva. Cuando una de las especies es invasora, el sistema ofrece una oportunidad interesante para entender los mecanismos ecológicos en la dinámica poblacional de especies invasoras. Dos especies de viudas negras coexistieron por más de 175 años en una localidad, una especie se considera invasora y la otra desapareció en años recientes. Se usaron variables ecológicas y análisis molecular de contenido estomacal en estas dos especies simpátricas para estudiar partición de nicho entre estas especies simpátricas. Se estimó la dieta por observación directa de remanentes de presas y, por primera vez en este género, por medio del análisis metagenómico de contenido estomacal. Se encontró solapamiento de nicho significativo entre las dos especies para variables de microhábitat y dieta. Usando metagenómica en los contenidos estomacales de las arañas, se recuperó una media de reads de 19,28% en
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33 especímenes de Latrodectus sp. y 19 especímenes de L. geometricus. Nuestros resultados muestran una recuperación de presas de 15,55% para Latrodectus sp. del total de los reads obtenidos, el 84,45% pertenece a L. geometricus. Las diversidades alfa calculadas para la comunidad de presas son muy similares para las dos especies, a pesar de la diferencia en reads de presa recuperados, incluso a niveles taxonómicos bajos. Se esperaban diferencias en la composición de nicho entre las dos especies simpátricas, pero los resultados soportan un solapamiento de nicho completo.
Palabras clave: Competencia, segregación, NGS, Latrodectus.
Introduction
Species interactions are often represented as a complicated network of relationships, an interaction network or a food web, which helps in the analysis of ecological disturbance, niche differentiation, and the co-existence of species in a community (Spiller and Schoener 1994, Bulleri et al. 2016). The species interactions are in turn affected by species’ response to environmental conditions such as temperature, humidity, and food availability, often summarized under the concept of niche (Pianka
1974, Holt 1987). Sympatric species are expected to undergo niche partitioning due to the ecological advantages of reducing competition for the same resources (Hardin 1960,
Holt 1987).
Interspecific competition plays an important role in the structuring of biological communities and thereby affects the probability of settling by invasive species (Hann
1990). When invasive species resemble any local species in ecologically relevant traits such as body size, consumption rate, prey use, and reproductive capacity, the invasive species may allegedly exhibit an ecological advantage that probably leads to the local extinction of the outcompeted native species (Hann 1990). Other factors may affect the
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outcome of these interactions, namely predators, parasitoids or abiotic factors that differentially affect the density of the interacting species (Hann 1990).
Incipient species may arise in the context of niche partitioning, due to the effect of natural selection on ecologically relevant traits that reduce interspecific competition
(Hardin 1960, Wiens 2004). Species tend to retain similar ecological niches over evolutionary time, and they often fail to adapt to new environmental conditions; thus, factors promoting the occupancy of new niches have the potential to promote the creation of new lineages (Wiens 2004). In this view, ecological specialization limits the habitat breadth and the evolutionary potential of a given species (Gruner 2007). Summing up, competitive interactions arising during the arrival of an invasive species may lead to local extinction of ecologically similar species, to adaptations allowing niche differentiation, and eventually to profound evolutionary divergence (and perhaps speciation) when any of the species ends occupying fundamentally new niches. The actual outcome of each interaction may actually depend on the intensity of resource sharing (Levin 1970, Cushing et al. 2004).
Web-builder spiders are considered generalist predators (Salomon 2011) and the high rate of prey consumption may increase the ecological dominance of one species over the other (Lewis 2013). We became aware of the sympatry between black widow species in Colombia, where two species, Latrodectus geometricus Koch, 1841, and Latrodectus sp., have shared habitat for more than 175 years in the Tatacoa region, according to medical articles and historical essays of the region (Koch 1841; Aguirre-Plata 1914;
Hamburger 1938; Gilij 1955; Boussingault 1985). One of them, L. geometricus is a widely distributed species and may have played the role of invasive species at some point.
Although the species was described in 1841 by L. Koch, and the holotype is from the
Tatacoa desert in Colombia (Vincent et al. 2008, Marie and Vetter 2015), the origin is
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thought to be African (Garb et al. 2004; Marie and Vetter 2015). This species is the only cosmopolitan black widow, showing great adaptability to different ecosystems. There are populations reported in the United States (including Hawaii), Jamaica, Bermuda, Haiti,
Cuba, Israel, Turkey, India, Australia, Papua New Guinea, Indonesia, the Philippines and
Japan (Vetter et al. 2012), and the species has been described as invasive (Garb et al.
2004; Vincent et al. 2008; Taucare-Rios 2011; Rueda 2012; Vetter et al. 2012; Lewis
2013). The species is also very well adapted to anthropogenic constructions, spins a funnel for hiding and the egg sacs show a characteristic pointy structure (Taucare-Rios
2011; Vetter et al. 2012). Latrodectus sp. is being also described from the Tatacoa region.
The co-existence of L. geometricus and Latrodectus sp. in Tatacoa has taken place for more than 175 years, according to reports, species descriptions, and medical articles that describe a red venomous spider in the Tatacoa desert since 1538 (Aguirre-Plata 1914;
Hamburger 1938; Gilij 1955; Quintana and Otero 2002; Boussingault 1985).
The diet of Latrodectus spiders has been studied by direct observation in British
Columbia (Salomon 2011), the United States (Nyffeler et al. 1988), Spain (Hódar and
Sánchez‐Piñero 2002), and Argentina (Pompozzi et al. 2013). The results of these studies corroborate that black widow spiders are generalist predators with preferences depending on the prey availability in each ecosystem (Mayntz et al. 2005).
This study aims at evaluating the niche overlap between L. geometricus and L. garbae in the Tatacoa desert. Our central hypothesis states that the species partition their niches in at least some of the studied dimensions. Dietary niche breadth is calculated for each species using direct-observation and metagenomic data. Molecular techniques have been applied for phylogenetic and evolutionary studies of Latrodectus (Garb et al. 2004;
Garb and Hayashi 2013; Haney et al. 2014). However, this is the first study to our knowledge that uses the metagenomic approach to gut content analysis in Latrodectus.
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Methods
The study was conducted in the Tatacoa “desert”, a dry forest located north of the
Departament of Huila, Colombia. In total, 749 webs of the two sympatric species were measured and included in the analysis. Sampling and measurements took place three times each year, during 2014, 2015 and September 2016 following the same fortuitous transects (Figure S1). Measurements stopped after 2016, when Latrodectus sp. was no longer found in the ecosystem, no systematic decline in the population density was recorded in the previous samplings and no specimens were found in later field trips. All specimens were collected under collection permits emitted by the Colombian Authority of Environmental Licenses ANLA.
To compare microhabitat use and web attributes, eight variables were measured: environmental temperature, relative humidity, internal (where the spider retreat is located) and external substrate temperature (substrate exposed to direct sun light), web length, width and height, specimen temperature and total body length. In each web measured, number of males, egg sacs and juveniles were noted. Species associated to the egg sacs and the presence of males were also noted, as it was common to find heterospecific samples in a determined web. Ecological measurements were only taken if the female was found in the web. Methods standardization in the field, was accomplished during 2013. Measurements were taken from early morning, until the environmental temperature started to be lower than external substrate temperature in the afternoon; when this change happened, measurements were stopped for the day.
Hypervolume simulation: Niche hypervolume was calculated using the R package “Hypervolume” (Blonder et al. 2014). All ecological variables were normalized to z-scores to have comparable dimensions. Gaussian kernel density estimation was used
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by the estimation of kernel bandwidth for each dimension included in the simulation, with a quantile probability requested of 0.95.
Diet by direct observation of prey cocoons: Latrodectus spiders lack the basal grooves with teeth on its margins in the cheliceral fang, so the prey is left as an empty cocoon that can be identified by direct observation (Foelix 2011). To estimate dietary niche by direct observation, 167 webs were analyzed, with a total of 483 prey cocoons identified. Dry cocoons were collected in paper envelopes; prey that were still being consumed were kept in individual vials with 70% alcohol. Samples were taken to the
Zoology and Aquatic Entomology Laboratory for identification to the lowest taxonomic rank possible.
Gut content analysis using Next Generation Sequencing: We also used metagenomic analysis to estimate dietary niche overlap between the species. Molecular gut-content analysis allows the detection of arthropod predation. Most results are qualitative, yet still sufficient to make ecological inferences. For example, molecular gut content analysis has been used to estimate dietary niche breadth and prey selectivity in a given ecosystem, to show that scavenging and secondary predation are common in nature, to prove that cannibalism makes a significant contribution to prey population suppression, and to elucidate the relationships making up a food web in complex invertebrate communities (Pompanon et al. 2012; Greenstone et al. 2014; Pinol et al. 2014).
Eighty specimens were collected (35 females and 5 males of each species) in
Natagaima, Tolima, Colombia (3°37'36.0"N 75°05'39.5"W). Each specimen was placed in an individual vial with 99% ethanol to avoid cross contamination. The spiders were transported to the Ecology and Aquatic Entomology Laboratory in Bogotá, Colombia, and were stored at -20°C until used. All NGS analysis were made at Evolab, University of California, Berkeley, CA, USA. Digital pictures were taken on a backdrop of graph
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paper to keep as voucher, and to make measurements of the cephalothorax to use as a metric for spider body size (Hagstrum 1971). All samples were deposited in the National
History Museum at Universidad de los Andes, in the invertebrate collection.
The abdomen of each specimen was removed and cut in half through the longitudinal axis with a sterile razor blade. The samples were placed in an extraction plate with two stainless steel beads of 3 mm in diameter (Qiagen, USA) in each well. A
GenoGrinder (SPEX SamplePrep, LLC) was used to grind the tissue for two 3-minute rounds at 1300 Hz. DNA was extracted using the Qiagen Puregene kit (Qiagen, USA) following the manufacturer’s protocol. The low molecular weight portion of the extracted
DNA was recovered using a mix of 0.75X Ampure Beads XP (Beckman Coulter, USA), according to the protocol of Krehenwinkel et al. (2017).
PCR was performed using the primer pair ZBJ-ArtF1c / ZBJ-ArtR2c (Zeale et al.
2011), which has proven to be effective for gut content analysis in arthropods and shows low bias in amplification (Krehenwinkel et al. 2017). Also, the primer amplifies a small fragment (~200 bp) of the mitochondrial COI gene, which works well for gut content analysis because the DNA of the prey is expected to be digested or degraded down to short fragments (Pompanon et al. 2012; Krehenwinkel et al. 2017). Indexing PCR and library preparation were conducted as described in Krehenwinkel et al. (2017). The libraries were sequenced on one flow cell of the Illumina MiSeq using V2 chemistry and
150 bp paired end reads (Illumina San Diego).
Sequences were demultiplexed by specimen and assembled using PEAR (Zhang et al.
2013), with a minimum overlap of 50bp and minimum quality of 30. After assembling, the sequences that showed more than 90% of bases with a Q30 were filtered and transformed into fasta files with FastX Toolkit (Gordon and Hannon 2010). Primers were trimmed and an OTU clustering was conducted with a similarity threshold of 95%, using
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USEARCH (Edgar 2010). Blastn (Altschul et al. 1990) was used to assign taxonomical identities based on the sequences deposited in GenBank. All non-arthropod and spider sequences were excluded.
Overlap of dietary niche: A matrix was built with the information obtained in both analyses: Direct Observation (DO) and metagenomic data (NGS) and scaled to Z- units. The numbers of prey taxa (and the proportions of prey) recovered for each analysis were compared. Analyses were conducted at the Order (133 webs) and Family (74 webs) taxonomic levels because of restrictions imposed by incomplete prey remains and short fragment length. We then conducted several analyses. The alpha diversity was calculated for each method separately and then compared to determine whether the two methods rendered similar diversity estimates. We used the R package (RCoreTeam 2016) Vegan
(Oksanen et al. 2007) and PAST 3 (Hammer et al. 2001) to calculate the Shannon Index as a measure of Alpha diversity. We compared the prey communities recovered by each method at the two taxonomic levels: Order and Family. Frequencies were calculated for each individual, i.e. each web analyzed. We expected to see an increase in alpha diversity as the taxonomic level got lower, because the prey recovery table was expected to become larger and more complex.
Results
During 2014 and 2015 the sampling frequencies were stable for the two species, with a mean sampling density higher for L. geometricus (0.79) compared to Latrodectus sp. of 0.21 (Table 1, Figure 1). Since 2016, Latrodectus sp. was not found any more in
Tatacoa, the search continued until 2017. Sampling density did not show a systematic decline in Latrodectus sp. population. New populations of Latrodectus sp. were found in dry ecosystems, approximately 67 km north of the desert, near the city of Natagaima,
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species identity of the new populations was confirmed by DNA analysis (Rueda et al. in press) The habitats were similar to Tatacoa, but the sampling density was inversed between L. geometricus and Latrodectus sp.
Webs were found in places where evident hiding spots were available. Some spiders were found hunting, hanging in the middle of the web at midday, despite the high temperatures and strong winds. Most of the webs were found on gray sedimentary rocks, followed by red igneous rocks. Other sampling substrates included sandy formations on the ground, small dry forest plants, tall grass, and occasionally under dry cow faeces. No significant difference was detected between species (Figure 1) in the use of substrates for web building (Wilcoxon signed ranks test; W = 20, p-value = 0.15).
Latrodectus geometricus built webs at 33.05± 28.15 cm above the ground (x ± sd), with a maximum height measured of 220 cm, while Latrodectus sp. showed a maximum web height of 62 cm from the ground, with an average of 17.92 ± 10.67 cm
(W = 39415, p-value = 2.641e-11). The length and width of the web also showed significant differences between the two species, with L. geometricus showed a mean length of 53.10 ± 30.17 cm and mean width of 49.53 ± 28.32 cm, and Latrodectus sp. had a mean length of 44.13 ± 22.97 cm and mean width of 40.59 ± 21.81 cm (Figure 2, Table
S1) (Length: W = 31974, p-value = 0.02432; width: W = 32299, p-value = 0.02).
Relative humidity (95% CI: L. geometricus 35.70-37.06%; Latrodectus sp. 34.45-
37.32%), environmental temperature (95% CI: L. geometricus 36.52-37.14°C;
Latrodectus sp. 36.44-37.65°C) and external substrate temperature (95% CI: L. geometricus 35.70-37.06%; Latrodectus sp. 34.45-37.32) measured in each web showed no differences between the two species (Figure 3-4, Table S1) (Relative humidity: W =
30416, p-value = 0.18; Environmental T°: W = 29066, p-value = 0.59; Ext. substrate T°:
W = 26824, p-value = 0.42). Internal substrate temperature (95% CI: L. geometricus
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36.06-36.90°C; Latrodectus sp. 37.40-39.43°C) showed a significant effect on body temperature (F(1,726)= 1954.19, MSE=15928.7, p< 2e-16), but no differences between species (F(1,726)= 3.68, MSE=30.0, p=0.06) with a mean in L. geometricus of 36.48 ±
5.22 °C and for Latrodectus sp. a mean of 38.41 ± 6.20 °C (Figure 4).
Latrodectus geometricus females were bigger (8.41 ± 3.42 mm), than Latrodectus sp. (8.00 ± 2.45 mm) and also produced more egg sacs (1.14 ± 1.64, maximum 11;
Latrodectus sp. 0.32 ± 0.57, maximum 3). Heterospecific egg sacs were often found in the web of a female, and the frequency was similar in Latrodectus sp. (0.06 ± 0.26) compared to L. geometricus (0.03 ± 0.21). The number of males on a web was much higher in L. geometricus (0.44 ± 0.87, maximum 3) than in Latrodectus sp. (0.12 ± 0.35, maximum 2). The presence of heterospecific males was only slightly higher for
Latrodectus sp. (0.17 ± 0.49 vs 0.04 ± 0.41). For complete information in ecological variables see Table S1.
The niche hypervolume was calculated using the R package “Hypervolume”
(Blonder et al. 2014). All ecological variables were normalized to z-scores to have comparable dimensions, and Gaussian kernel density estimation was used. All hypervolume analysis data are thus given in z-scores. Latrodectus geometricus hypervolume was 256.59, with a random point density of 370.53 and a number of random points of 95075, the kernel bandwidth estimated was of 0.31 for all 5 dimensions, with a quantile probability requested of 0.95. Latrodectus sp. hypervolume showed a total volume of 347.02, with a random point density of 289.06 and a number of random points of 100307, the kernel bandwidth estimated was of 0.41 for all 5 dimensions with a quantile probability requested of 0.95. Figure 5 shows all the dimensions for both species and the observed overlap. The hypervolume of Latrodectus sp. appears to be bigger than the hypervolume for L. geometricus, because some of the points are outside the main
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boundary, building up a hypervolume with spaces. The main hypervolumes are completely overlapped for both species and the niche for L. geometricus is bigger than the niche simulated for Latrodectus sp.
Variable importance was calculated for both hypervolumes and for both species the most important dimension is environmental temperature (z-scores: L. geometricus:
2.15; Latrodectus sp.: 2.45) followed by relative humidity (z-scores: L. geometricus:
2.12; Latrodectus sp.: 2.29), and external substrate temperature (z-scores: L. geometricus:
1.99; Latrodectus sp.: 2.27). The least important variable for the niche hypervolume in L. geometricus is internal substrate temperature (z-score: 1.89) and for Latrodectus sp. is spider body temperature (z-score: 2.02).
The Euclidean distance between the centroids of each hypervolume is of 0.10 and the minimum Euclidean distance between the random points comprising either hypervolume is of 0.15. The overlap between the hypervolumes of L. geometricus and
Latrodectus sp. with the Jaccard similarity index (“volume of intersection of 1 and 2 divided by volume of union of 1 and 2”, Blonder et al. 2014) is 0.49, and the Sorensen similarity index (“twice the volume of intersection of 1 and 2 divided by volume of 1 plus volume of 2”, Blonder et al. 2014) is of 0.66. The union between the two hypervolumes is of 403.85 with a unique fraction of the hypervolume for L. geometricus relative to
Latrodectus sp. of 56.83, and a unique fraction of hypervolume of Latrodectus sp. relative to the hypervolume of L. geometricus of 147.26.
In L. geometricus the niche is wider for all variables measured and there is a complete overlap with Latrodectus sp., but the niche hypervolume calculated for
Latrodectus sp. has a bigger volume because of the many holes it has. The points for the simulation created holes in the main boundary showing it with a wider volume, but the
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main hypervolume is small in shape. In Figure 5, those boundaries can be seen as well as the main volume completely overlapped between the two species.
Trophic niche: The determination and analysis of trophic niche by direct observation (DO) was accomplished with the identification of 483 prey items from 133 webs, distributed in 11 orders and 28 families. Many samples were recovered in good shape, but most of them were crushed and in small pieces. The strong winds and rain can affect and destroy the remains. Also, ants were seen “stealing” the prey after the spider had finished eating and returned to its retreat; this can also affect the recovery of a comprehensive sample to describe diet in arthropods by direct observation. Prey items belonging to all orders recovered (11) were identified in the diet of L. geometricus (For a complete list see Table S2). Latrodectus sp. only had prey items belonging to 7 Orders.
Morisita overlap index was calculated using R package “spa” (Zhang and Zhang 2013) with a value of 0.91 for Order and 0.86 for Family (Krebs 2014). This index goes from 0 to 1, 0 meaning no overlap and 1 complete trophic niche overlap.
Sequencing of gut content, following the enrichment protocol of prey DNA, recovered a mean of 19.28% of prey reads for all samples; the remaining reads corresponded to spiders and were eliminated. 40% of the samples showed a prey recovery of less than 10%. Samples were distributed in 6 Orders and 28 Families. All orders recovered by NGS method were also recovered by DO: Coleoptera, Diptera, Hemiptera,
Hymenoptera, Lepidoptera and Orthoptera. Although the number of orders recovered by both methods is high, no differences were found between them. At a lower taxonomic level, family, only 94 webs and 336 prey items were retained for direct observation to avoid mistakes with not identifiable samples. Only four families were recovered by both methods: Acriididae, Carabidae, Gryllidae and Tenebrionidae.
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Diversity indexes were calculated for both methods and both species with 10000 bootstraps using Past (Hammer et al. 2001); a Diversity t test was also conducted for each method. Order taxonomic rank for DO showed a Shannon Index for L. geometricus of
1.55 and for Latrodectus sp., 1.26 (Figure 6). Indexes for the NGS method were 1.003 for
L. geometricus and 1.21 for Latrodectus sp. Indexes obtained showed a significant difference between both species in both methods (DO: t(329.5) = 3.7, p = 0.0003, NGS: t(55909) = -4704, p = 0), being higher in L. geometricus for DO, and in Latrodectus sp. for NGS. For DO, 11 orders were found and only 7 were shared for both species: Araneae,
Coleptera, Hemiptera, Hymenoptera, Orthoptera, Scorpionida and Scutigeromorpha. The other 4 were only recovered for prey items from webs of L. geometricus: Blattodea,
Diptera, Lepidoptera and Neuroptera. For NGS analysis, of the 6 orders recovered:
Coleoptera, Diptera, Hemiptera, Hymenoptera, Lepidoptera, and Orthoptera, only one is not shared in both species: Orthoptera that was only recovered for L. geometricus. Also, the Shannon index is higher for Latrodectus sp. For a complete list of prey remains and reads recovered see Table S1.
For Family level or lower taxonomic ranks, using Direct observation, the Shannon
Index for L. geometricus was of 2.62 and for Latrodectus sp. was of 2.27. Indices for
NGS method were of 1.73 for L. geometricus and of 1.65 for Latrodectus sp. (Figure 7).
Indices obtained showed a significant difference between both species in both methods
(DO: t(103.18) = 1.59, p = 0.011; NGS: t(43413) = 16.69, p = 2.64e-62), being higher in
L. geometricus for both methods. The next taxonomic groups were recovered for both species in the DO method: Acrididae, Apidae, Tribe Atinii, Buthidae, Carbidae,
Subfamily Cicindalinae, Unidentified Coleoptera, Cydnidae, Elateridae, Formicidae,
Gryllidae, genus Latrodectus, Subfamily Scarabeinae, and Sphecidae. In the NGS method we found families that were not shared by the two species: Acrididae, Chironomidae,
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Cosmopterigidae, Dryinidae, Gryllidae, Muscidae, Oecophoridae, Sciaridae, and
Vespidae, were only recovered for L. geometricus, while Carabidae, Cixiidae,
Geometridae, Gracillariidae, Melyridae, Miridae, and Pipunculidae, were only recovered for Latrodectus sp. To see the complete table of families, see Table S2.
Discussion
We expected differences in niche composition between the two sympatric species, but results support a complete overlap. Dietary niche size of these generalist predators can be constrained by prey availability, ecological functions, and developmental stages.
Thus, in order to understand variability in dietary niche size, it is important to understand the ecosystemic function; the trophic network and how relations with other species can change the amount and type of prey available to each group (Pinol et al. 2014). Some of the traits considered to be important in trophic niche separation in spiders are body size
(Arim et al. 2010), hunting mode and microhabitat; because they determine the frequency of encounter and the type of prey the spiders can feed on. For example, if a spider reaches a bigger size, it can eat bigger prey (Sanders et al. 2015).
Competition plays an important role in community structure, but this trophic specialization is not the only factor that controls species co-existence; it is also the presence of other top predators and abiotic factors that determine the density of each population (Hann 1990). When an invasion takes place, the invasive species has the survival advantage, even if the foraging success is the same for both; because other factors
(predators and abiotic) are not affecting the abundance of the invasive species (Hann
1990).
In our study, L. geometricus is considered invasive because the closest phylogenetic relative is African (Marie and Vetter 2015), while Latrodectus sp. is an
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endemic species. After 2016, Latrodectus sp. Tatacoa population disapeared, and we found enough reasons to prove competition and segregation due to the presence of L. geometricus. Ecological niche is wider in the invaside species. No significative difference was found in any ecological variable between species, but specimens of Latrodectus sp. were found in more humid and warmer places than L. geometricus. This can be an explanation to the outlyers in the hypervolume simulation that makes the niche volume of Latrodectus sp. wider than the niche of L. geometricus; the simulations do not create a homogeneous volume, the package recognizes holes in the niche and adds them to the simulation (Blonder et al. 2014). Data for the 3 years of sampling was used, and Figure 5 shows the niche overlap for each of the five ecological dimensions used in the simulation between the two species studied. Competition for these resources, space and prey was taking place before the disappearance of Latrodectus sp. was recorded.
Also, L. geometricus showed more egg sacs in each web, with a maximum of 11 against 3 for Latrodectus sp.; male presence showed a significative different as well, with a max of 8 for L. geometricus and 2 for Latrodectus sp. This shows that L. geometricus has a higher reproductive rate and can have an ecological advantage over the native species in the Tatacoa ecosystem with a faster reproduction rate.
Trophic niche by direct observation proved to be a hard and time consuming job, and quantification of diet using identification of prey remains can give biased results. As observed in the field experiments, wind, rain, and other animals interactions (e.g. ants) can influence the prey remains in the webs; also, spiders were seen cutting the threads after eating, so prey would fall to the ground.
It is important to note that genomic studies of arthropods in Colombia are just beginning, and the sequences available for comparison in GenBank are thus limited leading to misidentifications. Also, the sequences we are amplifying are short, so two
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different genera can be mixed up due to sharing that specific sequence in different positions of the COI gene. Studies of animal diversity in the dry forests of Colombia are restricted to birds (Hilty and Brown 1986), lizards (Prado and Gibbs 1993), and scorpions
(Álvarez et al. 2013). Thus, the poor knowledge of these ecosystems opens the need of building up a reference library in order to have coherent results when using NGS for gut content analysis of generalist predators. Because the current COI databases are not exhaustive, only a more or less distant relative of the actual prey taxon is usually identified using database searches (Pompanon et al. 2012). Also, in tropical forests and other biodiversity hot-spots, NGS cannot identify all prey species by DNA sequences; there is a need to build a library for each ecosystem in order to determine with high exactitude the components of any specific diet (Symondson and Harwood 2014). Thus, it is plausible that the best hits for two different sections of the barcode region could end up being different species (Pompanon et al. 2012; Krehenwinkel et al. 2017). That is why at lower taxonomic levels the trophic niche is not the same between the two methods, DO and NGS.
However, even without that specific library, the niche breath and niche overlap can be calculated by molecular operational taxonomic units (MOTU) classified by family or order. So even if the prey is not identified the resource partitioning can be seen
(Symondson and Harwood 2014). Another limitation of NGS is that it does not allow to distinguish different life stages of prey groups (adult versus pupal form, etc.) which represents niche differentiation and can allow co-existence of predators in a given ecosystem (Clare 2014). Despite these shortcomings, NGS is still an exceptionally powerful tool to recover information on dietary niche of animals.
This NGS approach can be considered as a tool in conservation biology because it helps in vulnerability assessment, where niche competition and environmental change
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are involved in population decline, so it is important to determine niche breath and changes in it with abiotic variability (Clare 2014). This approach also helps in habitat restoration programs where it is important to determine the dietary requirements of the focal species so the environment can fulfill these ecological requirements (Clare 2014).
NGS helps with the identification of vulnerable ecosystems in need of conservation by identifying threatened species in lower or higher levels or establishing ecosystem pathways related with contamination (Clare 2014). Another important use of NGS is the analysis of the impact an invasive species has on a determined ecosystem, it gives information to evaluate vulnerable species and establish contingency plans to avoid extinctions (Clare 2014).
The size selection of low molecular weight DNA worked to enrich prey DNA, which is usually heavily degraded in the spider’s gut (Krehenwinkel et al. 2017), allowing us to recover 19% of prey reads. The prey recovered by NGS belongs mainly to
Hemiptera and Lepidoptera, species belonging to these orders usually have soft bodies and wings that can be lost by the action of wind or rain. DO identification of wings is impossible, leaving those prey remains in order taxonomic level. Also, the main prey identified by DO belongs to Coleptera and Hymenoptera, hard bodied animals that are easily recognizable and kept in the web, and very abundant in the ecosystem. Both methods recover a very similar prey composition at order taxonomic rank, while family gave very different results (Figure 8). These differences can be due to the lack of genetic information in the available prey items in that specific ecosystem, also, due to difficulties in the sampling and biases due to natural phenomena. There is a complete trophic niche overlap evidenced, both methods show different prey composition (restrictions related to each method) but both support the hypothesis of niche overlap and competition.
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The main results of this study show that there is a complete overlap in the environmental and trophic niche between L. geometricus and Latrodectus sp. L. geometricus’ origin is not determined yet, but it is thought to be from Africa, and it is considered invasive in all other ecosystems (Marie and Vetter 2015). In Colombia it may have outcompeted the native species Latrodectus sp. in the Tatacoa desert and caused a local extinction by competition. There are other variables that could affect the population density like parasitoids and predators that were not analyzed in this investigation.
Acknowledgments
We thank the Faculty of Sciences at the Universidad de los Andes for funding part of this investigation and field trips.
Conflict of Interest
The authors declare no conflicts of interest.
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Chapter 3 Figure legends
Figure 1. Left: Sampling frequency for each species of black widow spider during the ecological measurements in the Tatacoa desert. Right: Sampling frequencies of web encounters on different substrates for both black widow spiders in the Tatacoa desert.
Figure 2. Web maximum height (cm) vs web total length (cm) for both species of black widow spiders in the Tatacoa desert. Green: L. geometricus; red: Latrodectus sp.
Figure 3. Relative humidity and body temperature at the webs of two species of black widow spiders in the Tatacoa desert. Green: L. geometricus; red: Latrodectus sp.
Figure 4. Body temperature as a function of environmental temperature in two species of black widow spiders in the Tatacoa desert. Green: L. geometricus; red: Latrodectus sp.
Figure 5. Hypervolume created with scaled ecological variables for both species of black widow spiders, showing the three most important variables, body temperature: Tb, environmental temperature: Te, and Relative humidity: HR. Green: L. geometricus, Red:
Latrodectus sp.
Figure 6. Shannon index for diversity of prey order in two black widow spiders. Two methods of recovering prey information are also compared.
Figure 7. Shannon index for diversity of prey family in two black widow spiders. Two methods of recovering prey information are also compared.
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Figure 8. Trophic niche comparison at High taxonomic level (Order) between two species of black widow spiders. Other taxa include: Blattodea, Neuroptera, Scorpionida, and
Scutigeromorpha. These taxa represent 1.22% of the prey recovered with DO and 0% or the reads recovered with NGS for L. geometricus; and 1.29% of the prey recovered with
DO and 0% or the reads recovered with NGS for Latrodectus sp.
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Chapter 3 Table legends
Table 1. Sampling effort per field trip on two black widow spiders.
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Chapter 3 Figures
Figure 1.
Figure 2.
Figure 3.
141
Figure 4.
142
Figure 5.
Figure 6.
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Figure 7.
Figure 8.
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Chapter 3 Tables
Table 1.
Year Month Sampling days # webs L. # webs geometricus Latrodectus sp. 2014 April 7 237 60 July 3 66 24 October 3 41 11 2015 February 3 72 20 July 3 135 26 September 3 20 6 2016 September 2 60 0
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Supplementary Material Files: Chapter 3
Niche overlap in two sympatric species of black widows in Colombia
using ecological and genomic data
Authors: Alexandra Rueda 1,3*, Adolfo Amézquita2, Henrik Krehenwinkel3, Susan
Kennedy3, Rosemary Gillespie3, Emilio Realpe1
1 Laboratory of Zoology and Aquatic Entomology, Department of Biological Sciences,
Universidad de los Andes, AA 4976. Bogotá, Colombia.
2 Ecophysiology, behavior and herpetology Group, Department of Biological Sciences,
Universidad de los Andes, AA 4976. Bogotá, Colombia.
3 Environmental Sciences Policy and Management, University of California Berkeley
Mulford Hall, Berkeley, California, USA.
*Corresponding author [email protected]
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Figure S1. Map of the Tatacoa desert showing the sampling transects. Green: L.
geometricus, Red: Latrodectus sp. Map done with R and the package ggmaps (Kahle
and Wickham 2013)
Table S1. Micro-habitat variables summary. Mean, standard deviation, maximum, minimum and 95% confidence interval for each variable are shown.
Species Variable Mean sd Max Min 95% CI HR % 36.38 8.52 72 19.6 35.70 37.06 Environmental T°C (Te) 36.83 3.86 49.7 19.1 36.52 37.14 Body T°C (Tb) 36.77 5.27 54.1 23 36.35 37.20 Internal substrate T°C (Tsi) 36.48 5.22 63.8 23.8 36.06 36.90 L. External geometricus substrate T°C (Tse) 40.57 6.55 64.1 24.5 40.03 41.11 Web lenght (cm) 53.17 30.32 194 1.23 50.72 55.61 Web width (cm) 49.71 28.54 198 1.32 47.41 52.01 Web Maximum height 33.05 28.15 220 2 30.78 35.32 Web minimum height 16.52 22.88 155 0 14.66 18.37 147
Spider body lenght (mm) 8.37 2.15 19 2 8.19 8.55 Homo-especific Egg sacs 1.15 1.67 11 0 1.02 1.29 Hetero-especific Egg sacs 0.03 0.25 3 0 0.01 0.05 Homo-especific male 0.43 0.85 8 0 0.36 0.50 Hetero-especific male 0.04 0.41 9 0 0.00 0.07 Homo-especific juvenile 0.19 0.73 10 0 0.13 0.25 Hetero-especific juvenile 0.03 0.25 4 0 0.01 0.05 HR % 35.88 8.88 80.3 23.3 34.45 37.32 Environmental T°C (Te) 37.04 3.74 44.8 24.2 36.44 37.65 Body T°C (Tb) 38.33 5.80 53.7 23.4 37.39 39.28 Internal substrate T°C (Tsi) 38.41 6.20 57.6 25.8 37.40 39.43 External substrate T°C (Tse) 41.61 6.71 63.5 28.1 40.46 42.77 Web lenght (cm) 44.13 22.98 156 5 40.40 47.86 Web width (cm) 40.59 21.81 158 6 37.05 44.13 Web Maximum height 17.92 10.67 62 4 16.19 19.65 Latrodectus Web minimum sp. height 6.26 6.18 42 2 5.26 7.26 Spider body lenght (mm) 7.92 2.37 15 3 7.53 8.31 Homo-especific Egg sacs 0.33 0.57 3 0 0.23 0.42 Hetero-especific Egg sacs 0.06 0.26 2 0 0.01 0.10 Homo-especific male 0.12 0.35 2 0 0.07 0.18 Hetero-especific male 0.17 0.49 3 0 0.09 0.26 Homo-especific juvenile 0.11 0.46 3 0 0.02 0.19 Hetero-especific juvenile 0.03 0.18 1 0 0.00 0.06
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Table S2. Variables obtained in trophic niche determination for DO and NGS for both species at Order taxonomic level. For DO the numbers are the prey cocoons recovered and identified; for NGS the numbers are the prey reads for each taxon.
Direct Observation NGS (# reads) (prey items) Prey Order L. geometricus Latrodectus L. geometricus Latrodectus sp. sp. Araneae 20 5 - - Blattodea 1 0 - - Coleoptera 115 39 33354 1697 Diptera 2 0 726 934 Hemiptera 13 2 21512 11185 Hymenoptera 101 75 8508 4065 Lepidoptera 10 0 124075 16846 Neuroptera 1 0 - - Orthoptera 63 32 374 0 Scorpionida 1 1 - - Scutigeromorpha 1 1 - -
Table S3. Variables obtained in trophic niche determination for DO and NGS for both species at Family taxonomic level. For DO the numbers are the prey cocoons recovered and identified; for NGS the numbers are the prey reads for each taxon.
Direct Observation NGS (# reads) (prey items) Prey Family L. geometricus Latrodectus L. geometricus Latrodectus sp. sp. Acriididae 29 5 225 0 Apidae 2 2 - - Asilidae 1 0 - - Atini 27 12 - - Blattidae 1 0 - - Buthidae 0 1 - - Carabidae 0 1 0 239 Cecidomyiidae - - 10 791 Chironomidae - - 49 0 Cicadellidae - - 101 242 Cicindalinae 12 7 - - Cixiidae - - 0 1 Coleoptera 25 6 - - 149
Coreidae 1 0 - - Cosmopterigidae - - 32 0 Crysomelidae 6 0 - - Curculionidae 2 0 - - Cydnidae 0 1 - - Drosophilodae - - 581 25 Dryinidae - - 23 0 Elateridae 0 2 - - Erebidae - - 20311 8020 Formicidae 7 4 - - Geometridae - - 0 35 Gracillariidae - - 0 171 Gryllidae 14 8 149 0 Harpalinii 2 0 - - Hesperiidae - - 5772 1 Ichneumonidae - - 4 3517 Latrodectus 2 1 - - Lepidoptera 1 0 - - Lycaenidae - - 54538 21 Lycosidae 1 0 - - Melyridae - - 0 22 Miridae - - 0 25 Muscidae - - 15 0 Myrmeliotidae 1 0 - - Notodontidae - - 43374 8598 Oecophoridae - - 48 0 Oxyopidae 2 0 - - Pentatomidae 4 0 - - Pipunculidae - - 0 118 Psyllidae - - 21411 10917 Reduviidae 3 0 - - Scarabeinae 19 1 - - Sciaridae - - 71 0 Sphecidae 1 1 - - Staphylinidae - - 33339 1428 Tenebrionidae 1 0 15 8 Trechaleidae 1 0 - - Vespidae - - 3175 0
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Chapter 4:
Parasitoid habitat preference in Colombian black widow spiders
ALEXANDRA RUEDA 1*, ADOLFO AMÉZQUITA2, EMILIO REALPE1
Corresponding authors:
Alexandra Rueda ([email protected])
Running Title:
Latrodectus parasitoids
Cited references: 28
Figures: 4
Scope: Latrodectus ecology
Ready to Journal: Invertebrate Pathology
151
Parasitoid habitat preference in Colombian black widow spiders
ALEXANDRA RUEDA 1*, ADOLFO AMÉZQUITA2, EMILIO REALPE1
1 Laboratory of Zoology and Aquatic Entomology, Department of Biological Sciences,
Universidad de los Andes, AA 4976. Bogotá, Colombia.
2 Ecophysiology, behavior and herpetology Group, Department of Biological Sciences,
Universidad de los Andes, AA 4976. Bogotá, Colombia.
*Corresponding author: [email protected]
Abstract
This is the first approach to habitat preference in black widow’s parasitoids, and the first description of parasitoidism in Colombian Latrodectus. Parasitoids need a suitable environment in order to find a host and can act as ecological pressures in a community when co-existence of two possible hosts take place. The study was made in the Tatacoa desert, a dry forest southwest Colombia, where two species of Latrodectus co-existed until 2016. L. geometricus is considered invasive in many ecosystems and there are several studies of segregation of native black widows in the place of invasion. Latrodectus sp. was recently described for the country and local extinction was declared in the Tatacoa desert after 2016. Parasitoids were found in the egg sacs of both species, healthy egg sacs, and egg sacs with the presence of parasitoids were counted in each web and temperature was measured. Environmental variables like environmental temperature, relative humidity, and internal substrate temperature were also measured in each web. Results
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show that parasitoid wasps have a habitat preference and are more commonly found in humid and not so warm places. Also, a difference in egg sac temperature was noted.
Key words: Parasitoidism, Latrodectus, micro-habitat cues.
Resumen
Este es el primer estudio sobre preferencia de hábitat en parasitoides de viudas negras, y la primera descripción del parasitoidismo en Latrodectus en Colombia. Los parasitoides necesitan un hábitat adecuado para encontrar un huésped, y pueden actuar como presión ecológica en una comunidad cuando hay coexistencia de dos posibles hospederos. El estudio se realizó en el desierto de la Tatacoa, un bosque seco tropical ubicado al suroeste colombiano, donde dos especies de Latrodectus coexistieron hasta el 2016. L. geometricus se considera invasora en muchos ecosistemas y existen varios estudios sobre la segregación de las viudas negras nativas en el lugar de la invasión. Recientemente se describió Latrodectus sp. para el país y se declaró su extinción local en el desierto de la
Tatacoa después de 2016. Se encontraron parásitos en los sacos de huevos de ambas especies; se midió el número de sacos de huevos sanos, sacos de huevos con la presencia de parasitoides en cada red y la temperatura. Además, se tomaron variables ambientales como la temperatura ambiental, la humedad relativa y la temperatura interna del sustrato en cada tela analizada. Los resultados muestran que los parasitoides tienen una preferencia de hábitat y se encuentran más comúnmente en lugares húmedos y no tan cálidos. Además, se observó una diferencia en la temperatura de los sacos de huevos parasitados y sanos.
Palabras clave: Parasitoidismo, Latrodectus, señales de microhabitat
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Introduction
Successful parasitism begins with the appropriate recognition, and selection of host habitat (Vinson 1976; Godfray 1994), which in turns depends upon ecological and physiological cues related to host location (Godfray 1994). Parasitoids are also known to react to infochemical cues of the host habitat (Steidle and Reinhard 2003; Steidle and Van
Loon 2003; Segura et al. 2016). Allegedly, larvae or pupae memorizes the host they are consuming and the characteristics of the associated habitat; as adults, they will select this host over other possible hosts by habitat imprinting (Davis and Stamps 2004; König et al.
2015). Habitat selection can also be influenced by temperature, humidity, light intensity, wind, and habits of the parasitoids (Vinson 1976). Although there is extensive research on the chemical cues that parasitoids use to find the host, there is much less information on the effect of the physical factors on the behavior of wasp parasitoids (Steidle and
Reinhard 2003). Nevertheless, parasitoid’s behavior may play an important role in metapopulation dynamics and even shape the food web structure (Davis and Stamps
2004; Maunsell et al. 2015).
Parasitoids of black widow spiders (genus Latrodectus) have been described since
1942 (Bianchi 1945). They can be flies (Pseudogaurax signatus), wasps (Baeus latrodecti and Philolema), and mantispids (Bianchi 1945; Brambila and Evans 2001; Bibbs and
Buss 2011; Vetter et al. 2012). In the neotropics, the hymenopteran Pediobius pyrgo parasites egg sacs of L. geometricus (Schoeninger et al. 2015).
During the study of the ecological mechanisms allowing the coexistence of two
Latrodectus species in Tatacoa, Colombia, we became aware of the attack by parasitoids on the spider egg sacs. One of spider species even declined or eventually disappeared during the course of the study. Other studies show, that in sympatric spider populations, there can be other factors controlling co-existence, like predators, parasitoids or abiotic
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factors that keep the density below the ecosystem charge capacity (Hann 1990). We thus hypothesized that parasitoids exerts a differential effect on the spider species. The aims of this study were (1) to identify the parasitoid species found in the egg sacs of the two sympatric populations of Latrodectus in Tatacoa, (2) to test for difference between spider species in the prevalence and effects of parasitoids, and (3) to test for environmental correlates (environmental temperature, substrate temperature, and relative humidity) of parasitoid attack.
Methods
The study was conducted at the Tatacoa “dessert”, a dry forest located in the interandean valley of the Magdalena river (Colombia, 03°13’05.60’’N, 75’08’09.28’’W),
440 masl, and characterized by two rainy seasons in the year with less than 1000 annual mm of rain (Pizano et al. 2014). We collected egg sacs of black widow spiders only when a female spider (Latrodectus spp) could be seen in the web. We systematically inspected spider webs using transects prepared for a parallel study (Rueda et al. in prep.). The webs were rarely found above 50 cm from the ground and always anchored to rocks or ground vegetation (Rueda et al. 2017). The egg sac temperature was measured with an infrared thermometer in the spider web before being collected. We also measured environmental
(air) temperature, substrate temperature, and relative humidity. We further noted the number of egg sacs with parasitoids and the species identity of the spider. To test for environmental correlates of parasitoid presence, we compared the data collected and bootstrapped to complete a sample of 200 webs with parasitoids and 200 webs without parasitoids, using bootstrapping to calculate confidence intervals.
After microclimatic measurements, each encountered egg sac was collected with forceps and opened to evaluate the contents. When immature parasitoids were found, the
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egg sac was placed in a separate vial and kept in laboratory conditions until metamorphosis was reached. All other egg sacs were placed in a glass vial with absolute ethanol, and later kept at -20 ºC until used. All voucher of egg sacs, host specimens, and parasitoids were left in the Invertebrates Collection of Natural History Museum of
Universidad de los Andes in Bogotá, Colombia. Adult parasitoids were mounted and identified using a Leica EZ4HD stereoscope and following the identification key for the
Chalcidoidea super family. The identification was later confirmed by Dr Carlos
Sarmiento from the Laboratory of Systematics and Comparative Biology of Insects,
Universidad Nacional de Colombia.
Results
Non-attacked egg sacs show a wooly appearance and a white coloration that become yellowish over time; when the spiderlings are about to hatch, the middle of the egg sacs becomes dark. In contrast, the parasited egg sacs are more grey in the surface and the wooly appearance disappeared by the time of wasp hatching. In the early parasitoid stages, we detected no obvious difference in the egg sac appearance. In one egg sac from Latrodectus sp. we detected the simultaneous presence of two species of parasitoid wasps.
Two parasitoid wasp species were identified: Philolema latrodecti (Fullaway,
1953) (Eurytomidae) and Pediobius sp. Walker, 1846 (Eulophidae) (Figure 2). Of all inspected egg sacs, 150 showed the presence of parasitoid wasps and 56 were healthy.
The egg sacs with parasitoid wasps were colder (32.47°C (32.39-32.56), mean (95%CI)) than healthy egg sacs (33.53°C (33.47-33.6)), showing a significant difference (Wilcoxon signed ranks test; W = 36513, p-value < 2.2e-16). Also, attacked egg sacs revealed lower environmental temperature (36.67°C (36.63-36.7) vs 35.13 (35.03-35.22)), lower
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substrate temperature (35.38°C (35.32-35.43) vs 32.99°C (32.89-33.09)), and much higher relative humidity (39.57% (39.37-39.77) vs 35.09% (35.01-35.17)) (Figure 3). All of the environmental variables showed significative differences between them: W =
39558, p-value < 2.2e-16; W = 39928, p-value < 2.2e-16; W = 23, p-value < 2.2e-16; respectively.
Regarding the identity of spider species, L. geometricus had a larger number of egg sacs (2.17 ± 1.22, mean ± sd, maximum = 6) compared to Latrodectus sp. (1.38 ±
0.87, maximum = 4). Among them, 19% of egg sacs (60 out of 315 egg sacs from 251 webs) of L. geometricus and 30% (nine out of 30 egg sacs from 53 webs) of Latrodectus sp. were found with parasitoid wasps (Figure 4).
Discussion
At our study site, two species of black widows lived in sympatry for more than
175 years: L. geometricus and Latrodectus sp. (Gilij 1955; Boussingault 1985; Marie and
Vetter 2015). The latter could not be found, despite intensive search, after 2016 and is presumed to be locally extinct. The egg sacs of both species were found to be attacked by two species of parasitoids, which had already been reported for other species of
Latrodectus (Bibbs and Buss 2011; Vetter et al. 2012). Latrodectus behavior is characterized by sedentary females, that rest upside down in the web and rarely change the web location (Foelix 2011). It has been suggested that sedentary hosts are more often affected by parasitoids, because mobility and high dispersion rates makes host location difficult for the parasitoids and habitat cues play an important role in host- finding behavior (Gauld 1988).
Parasitoids have ecological requirements and enter levels of physiological stress, perhaps as much as the host, in extreme environments (Holt 1987; Godfray 1994). The
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first step after the emergence of a parasitoid is locating a suitable habitat and then a suitable host (Turlings et al. 1993), which may be accomplished by using chemical and physical cues related to the host habitat (Fatouros et al. 2008). It has been even proposed, that host location is a random process once the suitable environment has been found
(Vinson 1976). Species-specific parasitoids are also reportedly guided by physical and chemical cues that reduce the searching area (Vinson 1976).
Our results show that Latrodectus parasitoids are more often encountered in microhabitats with high relative humidity and low temperatures. Since we do not compare microhabitats with and without hosts, these results must be interpreted with caution.
Rather than showing the cues used by parasitoids to detect host microhabitats, our data show the conditions that render spider egg sacs more vulnerable to attack by parasitoids.
In this regard, they lead to the prediction that female spiders would benefit from building webs at very dry and hot microhabitats. If spider species differ in this behavior, they would also differ in their vulnerability to attack by parasitoids.
Latrodectus geometricus produced more egg sacs and were less often attacked by parasitoids than Latrodectus sp. but the difference is not significative (Figure 4). This comparison of parasitoid preference should be made with caution due to the low number of egg sacs recovered for the latter species. The data, however, should not be disregarded.
As described above, it is precisely Latrodectus sp. the species that completely disappeared at the study site. Our data are not only compatible with low reproductive potential and parasitoid attacks as potential causes of this extinction, they are also the only quantitative data available to date. Further studies at new localities are needed to further confirm this prediction.
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Acknowledgements
We thank the Biological Science Department, and the Science Faculty of
Universidad de los Andes for funding part of this project.
Conflict of Interest
The authors declare no conflicts of interest.
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Chapter 4 Figure legends
Figure 1. A: Pupae of parasitoid wasps (arrow) in an egg sac of the black widow spider
L. geometricus. B: Latrodectus sp. egg sac with parasitoid larvae and some Latrodectus eggs still intact (arrow). C: L. geometricus spider found inside the parasited egg sac with one of the adult parasitoid wasps next to it. D: Egg sac opening where the two adults
(spider and wasp) were found. Pictures by Alexandra Rueda.
Figure 2. Latrodectus parasitoids. Top: Philolema latrodecti, parasitoid wasp, found in the egg sacs of two Latrodectus species in Tatacoa. Left: Wasp male; picture by Emilio
Realpe. Right: L. geometricus egg sac with adult parasitoids on its surface after being opened in the laboratory with forceps. Picture by Alexandra Rueda. Bottom: Pediobius sp. wasp found in the egg sac of L. geometricus. Left: specimen, picture by Emilio Realpe.
Right: L. geometricus egg sac opened in the laboratory with adult parasitoid wasps and spiders, picture by Alexandra Rueda.
Figure 3. Environmental variables measured in each web of black widow spiders where egg sacs were found, according to the presence (YES) or absence (NO) of parasitoid wasps.
Figure 4. Left: Number of egg sacs found in the webs of each species. Right: Plot showing a binary model of parasitoid presence in each egg sac by spider species (R package yarrr (Phillips 2016)).
164
Chapter 4 Figures
Figure 1.
Figure 2.
165
Figure 3.
Figure 4.
166
General Conclusions
This thesis is an integrative approach to the study of ecological interactions, phylogenetics, biochemistry of a new black widow spider for the country. This investigation gives enough support to suggest that the “coya” spider from the Tatacoa desert, named in so many medical reports and historical essays, belong to a new
Latrodectus species. Along with the correct identification, separation from other species, phylogenetic location, and interespecific relations in the study case ecosystem, the spider is finally described with a scientific name and it’s ecology is studied further.
One of the arguments used to justify the new species hypothesis, is that none of the reported black widows for Colombia: L. geometricus and L. curacaviensis can produce the symptoms observed in the animal models tested in this investigation. Also, the SDS-PAGE experiment showed a band consistent with α-latrotoxin. These results oppened the possibility that the spider found in the Tatacoa desert belonged to a different, unidentified species. Also, the medical reports and historical essays talked about severe symptoms related to Latrodectism that can not be produced by any other Colombian spider.
Furthermore, we show enough genetic, morphological and geographic support to the hypothesis of a new species that could represent the “coya” spider. Also, we give enough support for the description of another candidate species for the department of
Santander, and we determine the synonimy between two Argentinian species. Both candidate species can be distinguished from other Colombian and worldwide species, because of color pattern, morphology, genetics, and geographical location. Latrodectus from Santander, shows an ontogenetic change in color pattern, very similar to L. hesperus but high genetic divergence. Latrodectus sp. from Tatacoa show close phylogenetical
167
relation to L. diaguita but reciprocal monophylyis obtained for both genes analyzed. New population reports are made for L. geometricus and L. curacaviensis. All species were genetically compared with the closest phylogenetic relative to give enough statistically validation to the new species hypothesis and the synonymy between L. diaguita and L. corallinus. Morphological description of male and female sexual organs showed little variation in this structures, consistent with what is reported in the literature.
While studying Latrodectus populations in Colombia, we found that sympatry is a common phenomena in this genus, and the most interesting case was found in the
Tatacoa desert, where L. geometricus and Latrodectus sp. co-existed for more than 175 years according to historical records. After 2016 Latrodectus sp. was not found and local extinction was declared. New populations of this species were found in the north of the
Tatacoa desert, where L. geometricus has not stablished yet.
The potential causes of this extinction could be related with inter-specific relations in a community that includes resource partitioning, parasitism, predators, among others.
Niche overlap was evaluated with ecological variables and molecular gut-content analysis and complete niche overlap was found between the two co-existing species. These results support the hypothesis of competition and can be one of the reasons Latrodectus sp. was segregated from the Tatacoa desert.
Also, two parasitoid wasps were found in Latrodectus egg sacs, in the Tatacoa desert. Parasitoid habitat preference was determined, showing more abundance in lower temperatures and higher relative humidity places, and host preference was not found.
Furthermore, L. geometricus has a higher reproductive rate, reducing the effect of parasitoidism on population density. Latrodectus sp., on the other hand, could have been under ecological pressure by parasitoidism due to the low reproductive rate and lower egg sac production.
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It is possible that the original population of Latrodectus sp. moved from the
Tatacoa desert to northern locations, in order to avoid resource competition with L. geometricus, and to be more protected from parasites. Other possible ecological pressures, such as predation, should be evaluated.
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Thesis Supplementary Material Files:
SYSTEMATICS, TOXINS, PARASITOIDS, AND NICHE: AN
INTEGRATIVE APPROACH TO THE STUDY OF A NEW
BLACK WIDOW SPIDER
Doctoral Thesis presented by:
Martha Alexandra Rueda Esteban
Advisor: Emilio Realpe PhD.
Co-Advisor: Adolfo Amézquita PhD.
Evaluator: Charles Griswold
Evaluator: Jorge Molina
170
Toxicon 125 (2017) 53e58
Contents lists available at ScienceDirect
Toxicon
journal homepage: www.elsevier.com/locate/toxicon
Toxicity evaluation and initial characterization of the venom of a Colombian Latrodectus sp.
* Alexandra Rueda a, , Emilio Realpe a, Alfredo Uribe b a Biological Sciences Department, Laboratory of Zoology and Aquatic Ecology LAZOEA, Universidad de los Andes, Bogota, Colombia b Biological Sciences Department, Biochemical Investigation Center CIBI, Universidad de los Andes, Bogota, Colombia article info abstract
Article history: The genus Latrodectus has not been studied in Colombia even though it is medically important world- Received 24 April 2016 wide; there are three species for the country, this study focused on a non-identified species found in the Received in revised form Tatacoa Desert in the Huila Department. This research is the first approximation to the extraction, 16 November 2016 composition analysis and toxicity evaluation of the venom of a species of the genus Latrodectus in Accepted 22 November 2016 Colombia; and aims to evaluate the toxicity by the initial characterization of its venom. The venom Available online 23 November 2016 extraction was accomplished with electrostimulation and total protein concentration was determined by the Lowry method and BCA assays from crude venom; with these methods, high protein concentration of Keywords: Latrodectus the samples was measured. Bioassays on mice were also made to evaluate the toxicity and compare the Spider symptoms produced by this Colombian spider to the Latrodectism Syndrome. Finally, an SDS-PAGE Venom electrophoresis was used to separate the main components of high molecular weight from the sam- SDS-PAGE ples and compared to a control of the venom of Latrodectus mactans to determine if the venom Black widow composition is different between these two species. Latrodectism © 2016 Elsevier Ltd. All rights reserved.
1. Introduction components, the a-Latrotoxin, is toxic to mammals. The bite of some species like Latrodectus mactans can be lethal (Quintana and Latrodectus (Walckenaer, 1805) is a spider genus that belongs to Otero, 2002; Rohou et al., 2007) but few cases are lethal if treat- Family Theridiidae (Sundeval, 1833). These spiders have a globular ment is supplied (Artaza et al., 1982). The symptoms presented abdomen, small body size, sexual dimorphism and show a wide after the bite of a black widow or close relatives like Steatoda is color pattern polymorphism in the abdomen, between and inside known as the Latrodectism syndrome (Haney et al., 2014). The first species. They live in dry habitats or near caves where the webs can symptoms that occur are local pain, sweating, itching, and edema. be found (Garb et al., 2004; Quintana and Otero, 2002). They are Systemic symptoms include fever, dizziness, cramps, spasms, dys- extremely calm, but they tend to bite when they are bothered or pnea, tachycardia, arrhythmia, among others (Duan et al., 2006; compressed (Artaza et al., 1982; Grisolia et al., 1992). Guerrero et al., 2010). These symptoms can last a few hours to The World Health Organization considers this genus dangerous more than a week and can lead to death because of pulmonary or (Quintana and Otero, 2002), and medically important because of cerebral edema or cardiovascular manifestations (Quintana and the severe clinical manifestations and possible lethality of the Otero, 2002). The symptoms also depend on the spider species, venom (Aguilera et al., 2009; Bonnet, 2004; Calvete et al., 2009). there are reports of differences in severity of the effect of the bite The venom contains peptides or protein fractions (Boeve et al., between species and among close related genera, like Steatoda and 1995) that act as toxins affecting the central nervous system of Parasteatoda (Garb and Hayashi, 2013). The median lethal dose in different organisms; these include latrotoxins and trigger a massive mice is already known for several species (Daly et al., 2001; Reyes- neurotransmitter release upon injection (Aguilera et al., 2009; Garb Lugo et al., 2009), these raise the question of whether this and Hayashi, 2013; Shukla and Broome, 2007). One of the venom Colombian species can be medically important. There are three species described in Colombia: L. curacaviensis (Müller, 1776) and L. geometricus (Koch, 1841) (Quintana and Otero, * Corresponding author. Cra. 1 No. 18A 12, Bogota, Colombia. 2002) and an unidentified species that is found in the Tatacoa E-mail addresses: [email protected] (A. Rueda), [email protected]. Desert, located in the State of Huila (Rueda et al., 2010). This last co (E. Realpe), [email protected] (A. Uribe). http://dx.doi.org/10.1016/j.toxicon.2016.11.255 0041-0101/© 2016 Elsevier Ltd. All rights reserved.