(DRAFT) - Taxonomy
Species GAMBUSIA, BIG BEND
Species Id ESIS252004
Date 14 MAR 96
TAXONOMY
NAME - GAMBUSIA, BIG BEND
OTHER COMMON NAMES - GAMBUSIA and BIG BEND
ELEMENT CODE -
CATEGORY - Fish
PHYLUM AND SUBPHYLUM - CHORDATA,
CLASS AND SUBCLASS - OSTEICHTHYES,
ORDER AND SUBORDER - ATHERINIFORMES,
FAMILY AND SUBFAMILY - POECILIIDAE,
GENUS AND SUBGENUS - GAMBUSIA,
SPECIES AND SSP - GAIGEI,
SCIENTIFIC NAME - GAMBUSIA GAIGEI
AUTHORITY -
TAXONOMY REFERENCES -
COMMENTS ON TAXONOMY -
Big Bend Gambusia
Gambusia gaigei Hubbs, 1929
KINGDOM: Animal GROUP: Fish
PHYLUM: Chordata CLASS: Osteichthyes
ORDER: Atheriniformes FAMILY: Poeciliidae
The Big Bend gambusia is a relatively plain, yellowish Poeciliid
up to 30 mm long, whose faint lateral stripe is the most pronounced
dark mark on the body. There is also a suborbital bar and a faint,
dark chin bar. The gonopodium has a pronounced elbow, with only one
or two segments.
The species was described in 1929 by Hubbs (01). The type
specimens are in the University of Michigan Museum of Zoology. Most
other specimens are in the Texas Natural History Collection.
Photographic illustrations of the Big Bend gambusia may be found in
(02) and a drawing in (15).
The species was considered as a senior synonym of two Mexican
species (Gambusia hurtadoi Hubbs and Springer, 1957; and G. alvarezi;
Hubbs and Springer, 1957) (03). This opinion was not accepted by many
authors (04,05,06,07,08,09,15). All authors agree that it is closely
related to G. senilis. It was placed in the G. nobilis species group
Taxonomy - 1 (DRAFT) - Taxonomy
Species GAMBUSIA, BIG BEND
Species Id ESIS252004
Date 14 MAR 96
by Hubbs and Springer (01,03) but that species group was divided, and
G. gaigei placed in the G. senilis species group by Rivas (04).
Subsequent authors have not recognized the G. senilis group as
distinct from the G. nobilis group (04,05) and place the Big Bend
gambusia in the G. nobilis group.
Taxonomy - 2 (DRAFT) - Status
Species GAMBUSIA, BIG BEND
Species Id ESIS252004
Date 14 MAR 96
STATUS
Coded Status
E: Federal Endangered
COMMENTS ON STATUS -
U.S. STATUSES AND LAWS:
The Big Bend gambusia (Gambusia gaigei) has been designated an
Endangered species pursuant to the Endangered Species Act of 1973
(50 CFR 17.11; P.L. 93-205, 87 Stat. 884; 16 U.S.C. 1531-1540),
as amended. The species has this status wherever found including
the State of Texas.
This species is protected by the Lacey Act (P.L. 97-79, as
amended; 16 U.S.C. 3371 et seq.) which makes it unlawful to import,
export, transport, sell, receive, acquire, or purchase any wild
animal (alive or dead including parts, products, eggs, or offspring):
(1) in interstate or foreign commerce if taken, possessed,
transported or sold in violation of any State law or
regulation; or
(2) if taken or possessed in violation of any U.S. law,
treaty, or regulation or in violation of Indian tribal law.
It is also unlawful to possess any wild animal (alive or dead
including parts, products, eggs, and offspring) within the U.S.
territorial or special maritime jurisdiction (as defined in
18 U.S.C. 7) that is taken, possessed, transported, or sold in
violation of any State law or regulation, foreign law, or Indian
tribal law.
RESPONSIBLE FEDERAL AGENCIES:
USFWS -Responsible for the management/recovery, listing, and
law enforcement/protection of this species.
NPS -Responsible for the law enforcement/protection of this
species with applicable State and Federal laws on
public lands under their control. Also responsible
for conservation (Nat. Park System Organic Act - 16
U.S.C. 1, 2-3)/management/recovery on National Park
Service lands. Taking, possessing, or disturbing of
Federally listed species is prohibited on NPS lands
(36 CFR 2.1, 2.2, and 2.3).
All Federal agencies have responsibility to ensure that any
action authorized, funded, or carried out by that agency is not likely
to jeopardize the continued existence of the species or result in the
destruction or adverse modification of Critical Habitat (50 CFR 402),
and to utilize their authorities to carry out programs for the
conservation of the species.
Status - 1 (DRAFT) - Status
Species GAMBUSIA, BIG BEND
Species Id ESIS252004
Date 14 MAR 96
STATE STATUSES AND LAWS:
STATE: Texas
DESIGNATED STATUS: Endangered
ADMINISTRATIVE AGENCY: Texas Parks and Wildlife Department
STATE STATUTE: 31 Texas Administrative Code Sec. 57.133
(127.30.003) July 1977
INTERNATIONAL STATUSES, TREATIES, AND AGREEMENTS:
The Big Bend gambusia is listed as Endangered in the IUCN Red
Data Book, Vol. 4, 1977; and is also listed by the Convention on
Nature Protection and Wildlife Preservation in the Western Hemisphere,
1970.
ECONOMIC STATUSES:
This species has value to naturalists and has a value as a
component of Texas' natural heritage of biotic diversity.
67/03/11:32 FR 04001/ - Listing as Endangered
79/05/21:44 FR 29565/29577 - Five year review
85/07/22:50 FR 29901/29909 - Five year review
Status - 2 HABITAT ASSOCIATIONS
HABITAT - AQUATIC
INLAND AQUATIC
LAND USE -
Shrub and Brush Rangeland
Streams and Canals
Reservoirs
NATIONAL WETLAND INVENTORY CODES
NWI NWICLS NWIMOD NWISPEC
Riverine, upper perennial AB1
Lacustrine, littoral AB1
Palustrine EM1
COMMENTS ON HABITAT ASSOCIATIONS -
The species can coexist with Gambusia affinis only in warm (30
degrees C plus) stenothermal flowing waters. Any alteration of
temperature or flow regimes will result in the elimination of the Big
Bend gambusia.
The Big Bend gambusia is known only from spring habitats of Texas
in the vicinity of Boquillas Crossing and Rio Grande Village in Big
Bend National Park (08).
Spring 1 and Spring 4 are important for sustaining Gambusia
gaigei in the Rio Grande Village area of Big Bend National Park.
These springs (about 270 meters apart) have been developed, altering
their natural flows. Chemical water analyses show them to have been
almost identical during the past 24 years and strongly suggest that
both springs have a common source. Springs 1 and 4 are intermediate
thermal springs, part of a cluster of six known thermal springs
concentrated in this area of the Big Bend of the Rio Grande.
Temperatures of the spring cluster are in the 30-50 degrees C range
with the warmest temperatures in Hot Springs and temperatures in
Springs 1 and 4 near 34 degrees C. The points of issue of Springs 1
and 4 are located along the contact of the upthrow side of the Santa
Elena Limestone of Cretaceous Age (13).
Thermal water issuing from both springs is of ancient origin with
the principal recharge zone located in higher elevations of the Sierra
del Carmen (13). The fault system, formed in Mesozoic times during
development of the Sierra del Carmen, serves both as a means for
collecting and recharging the groundwater system as well as an
avenue by which groundwaters are discharged in a spring line. In
addition to the concentrated flows of Springs 1 and 4, diffused
seepage occurs in the vicinity (08).
The natural habitat occupied by G. gaigei was clear, shallow
water fed by warm springs. Although the Big Bend gambusia will exist
in other habitats, the rapid replacement by mosquitofish (Gambusia
affinis) in eurythermal water suggests that the Big Bend gambusia is
best adapted to warm-spring areas. Other members of the Gambusia
nobilis species group in U.S. waters also are abundant in springs,
especially in those areas inhabited by mosquitofish downstream.
The species does best in dense aquatic vegetation (Chara is the
form in the refugium) (07).
Flooding based on flow regulation of the Presa Luis L. Leon in
Mexico (reservoir) could impact the species by allowing competitiors
and predators access to the species and habitat (08). Areas
immediately surrounding the habitats are used for recreational
Habitat Associations - 1 purposes (campgrounds) within the Big Bend National Park.
Little is known concerning the population biology and dynamics of
the Big Bend gambusia (Gambusia gaigei). Limiting factors are
considered to be the maintenance of stenothermal flowing spring water
(11).
The surrounding rangeland is classified as Texas savanna.
Habitat Associations - 2 (DRAFT) - Food Habits
Species GAMBUSIA, BIG BEND
Species Id ESIS252004
Date 14 MAR 96
FOOD HABITS
TROPHIC LEVEL -
CARNIVORE
LIFESTAGE FOOD FOOD PART
General Zooplankton
General Arthropods
General Worms
Food Habits - 1 (DRAFT) - Environment Associations
Species GAMBUSIA, BIG BEND
Species Id ESIS252004
Date 14 MAR 96
ENVIRONMENTAL ASSOCIATIONS
G = General A = Adult
LIM = Limiting RA = Resting Adult
J = Juvenile FA = Feeding Adult
RJ = Resting Juvenile BA = Breeding Adult
FJ = Feeding Juvenile P = Pupae
L = Larvae E = Egg
RL = Resting Larvae
FL = Feeding Larvae
LIFESTAGE ENVIRONMENTAL ASSOCIATIONS
G Aquatic Features: Springs [flowing]
Environment Associations - 1 (DRAFT) - Life History
Species GAMBUSIA, BIG BEND
Species Id ESIS252004
Date 14 MAR 96
LIFE HISTORY
FOOD HABITS:
Little is known about the food habits of the Big Bend gambusia
(Gambusia gaigei) (07). Presumably, as in other Poeciliids,
insect larvae and other invertebrates (zooplankton and worms) account
for most of the dietary intake of this species (07).
HOME RANGE/TERRITORY:
No information is known concerning territoriality but males are
presumed to be quasi-territorial (07).
PERIODICITY:
Unknown (07).
MIGRATION PATTERNS:
No migrations are known for the Big Bend gambusia (Gambusia
gaigei) (07).
COVER/SHELTER REQUIREMENTS:
The natural habitat occupied by G. gaigei was clear, shallow
water fed by warm springs (08). The species does best in dense
aquatic vegetation (Chara is the form in the refugium) (07).
REPRODUCTIVE SITE REQUIREMENTS:
Viviparous, no site requirements are known (07).
REPRODUCTIVE CHARACTERISTICS:
Little is known, but, brood numbers are dependent. Females
reproduce in June and August. Females of the closely related Gambusia
hurtadoi and G. alvarezi reproduce in January. This species is
presumed to be reproductive for 12 months. Fecundity is high in June
(up to 50 yg in small females) and low in August and January (up to 30
yg in large females) (14).
It is probable that, once inseminated, females can store sperm
for long periods, e.g., several months, and that interbrood intervals
in individual females is on the order of 1-2 months. These are
general features of reproduction in Gambusia species. Factors
limiting reproduction are unknown, but photoperiod, temperature, and
food availability all affect fecundity and interbrood intervals in
poeciliids (08).
PARENTAL CARE:
No parental care is known (07).
POPULATION BIOLOGY:
Little is known concerning the population biology and dynamics of
the Big Bend gambusia (Gambusia gaigei). Limiting factors are
considered to be the maintenance of stenothermal flowing spring water
(11).
SPECIES INTERRELATIONSHIPS:
Big Bend gambusia have been found associated with at least five
Life History - 1 (DRAFT) - Life History
Species GAMBUSIA, BIG BEND
Species Id ESIS252004
Date 14 MAR 96
other fish species within their native range. These included, at
Spring 4, two minnows (Notropis braytoni and N. lutrensis), another
poeciliid (mosquitofish, Gambusia affinis), an ictalurid (presumably
Channel catfish, Ictalurus punctatus) and a centrarchid (green
sunfish, Lepomis cyanellus). The channel catfish and green sunfish
are potential predators and were reported to have been purposefully
stocked into the overflow pool of Spring 4; perhaps the other species
accidentally were placed into the pool along with these game species
(02,10). These predators were removed and their impacts reportedly
were minimal. The two potentially competitive minnows also were
eliminated, but the mosquitofish survived and apparently is a serious
competitive threat to Big Bend gambusia (08). At least 18 other fish
species inhabit the Rio Grande adjacent to the habitats of the Big
Bend gambusia (16) but are not syntopic with the Big Bend gambusia.
They could move into the campground area in the event of a large-
magnitude flood.
Other species found with Big Bend gambusia include aquatic
invertebrates that may be prey items for G. gaigei, crayfish, a
variety of potential avian predators, and various plants including
bulrushes, cattails, saltcedars, and cottonwoods, which provide cover
and/or shade to the habitat (08).
The Big Bend gambusia (Gambusia gaigei) is unable to coexist with
Gambusia affinis in eurythermal waters, cool stenothermal waters, or
lentic warm stenothermal waters (03,08,11); however, G. gaigei can
coexist with Gambusia affinis only in warm (30 degrees C plus)
stenothermal flowing waters.
OTHER LIFE HISTORY DESCRIPTORS:
All documented natural occurrences of Big Bend gambusia are from
spring outflows. The two springs of natural occurrence differ
substantially in outflow temperature. Spring 4 has an outlet
temperature of 32-35.5 degrees C (13) and Boquillas Spring had a
temperature of about 23 degrees C on March 6, 1982. However, the fish
inhabiting Spring 4 did not live in the springhead itself, but
downstream where the temperature was influenced by ambient air
temperatures. Recorded water temperatures (summers of 1954-56) from
the Spring 4 outflow ditch, densely inhabited by Big Bend gambusia,
were cooler and near ambient. Recorded temperatures from the refugium
also fluctuate widely dependent upon air temperature and location
(08).
Life History - 2 (DRAFT) - Management Practices
Species GAMBUSIA, BIG BEND
Species Id ESIS252004
Date 14 MAR 96
MANAGEMENT PRACTICES
RESULT MANAGEMENT PRACTICE
Beneficial Restricting/regulating human disturbance of populations
Beneficial Maintaining/Controlling Water Flow
Beneficial Maintaining undisturbed/undeveloped areas
Beneficial Reforestation
Beneficial Stocking captive-reared wild-strain animals
Beneficial Controlling/Removing Exotic Vertebrates
Adverse
Existing
Adverse Low Gene Pool
Existing Low Gene Pool
Adverse Predation
Existing Predation
Adverse Recreational development
Existing Recreational development
Adverse Siltation
Existing Siltation
Adverse Water Temperature Alteration
Existing Water Temperature Alteration
Adverse Flooding
Existing Flooding
Adverse Groundwater drawdown
Existing Groundwater drawdown
Adverse Reservoirs
Existing Reservoirs
Adverse Dredging
Existing Dredging
Adverse Competition
Existing Competition
Adverse Exotic/Feral/Introducted Species
Existing Exotic/Feral/Introducted Species
COMMENTS ON MANAGEMENT PRACTICES -
The type locality (Boquillas Spring) population was extinct by
1954 as the spring was dry (03,08).
The Spring 4 population (Graham Ranch Warm Spring) declined
drasticlly between June 1954 and April 1956 (10). The construction of
a children's fishing pool for the proposed Rio Grande Village
Campground had drastically altered the springs flow regimes (11) and
fish from the Rio Grande, presumably including Gambusia affinis were
introduced into the Spring 4 system (10). Twenty-five G. gaigei were
removed by seine and transferred to live tanks prior to the Spring 4
system being treated with Rotenone (in October 1956) in a futile
effort to eradicate Gambusia affinis (08,10). Three individuals
eventually survived and constituted the entire stock available for
recovery actions. These individuals and offspring flourished in the
first refugium until Gambusia affinis inadvertantly gained access to
the refugium by 1960 (10). Another refugium based on Spring 1 water
was constructed and the population again flourished (10). Green
Management Practices - 1 (DRAFT) - Management Practices
Species GAMBUSIA, BIG BEND
Species Id ESIS252004
Date 14 MAR 96
sunfish (Lepomis cyanellus) were found in that refugium and
eliminated (12) in 1968. A mass mortality occurred during unusually
cold weather in December 1975 (13) and this difficulty was resolved by
establishing a flow through system so that Spring 1 temperatures (30
degrees C plus) would exist around the inflow (12). The overflow
ditch subsequently supported a small population at most times (08).
In 1984 following the recommendations of the Recovery Plan (08), fish
were released into the Spring 4 outflow and a sparse population was
located in stenothermal spring water, in a once dry marsh, 50 meters
southwest of Spring 4 (11).
It is considered that Gambusia gaigei cannot coexist with
Gambusia affinis (08); the existence of a population coexisting in
stenothermal flowing spring water (11) suggests that the Big Bend
gambusia can coexist with congenera in warm stenothermal spring water
(13).
The primary threats are potential declining spring flow through
groundwater drawdown or diversion for additonal park development,
changes of outflow temperature, and excessive abundance of Gambusia
affinis. Surface runoff and flooding of the Rio Grande River are also
threats. Periods of unusually high precipitation could result in
surface runoff causing silt deposition and/or erosion in the refugium
or spring runs. Flooding or broad surface runoff could provide an
avenue for invasion of G. affinis or other undesirable species. The
flooding threat is based on the regulation of flow from Presa Luis L.
Leon into the Rio Conchos, Mexico (08).
APPROVED PLAN:
U.S. Fish and Wildlife Service. 1984. Big Bend Gambusia Recovery
Plan. U.S. Fish and Wildlife Service, Albuquerque, New Mexico.
43 pp.
The goal of the Recovery Plan is to assure the survival of the
Big Bend gambusia (Gambusia gaigei) through improvement of its status.
It is important to understand the biology and ecology of the species
so that in the event of a decline, causative factors can be identified
and remedied as quickly as possible. The plan stresses the importance
of understanding:
1) Competition with mosquito fish (Gambusia affinis).
2) Gambusia gaigei's food preferences and the biology of their prey.
3) This species' reproductive variables.
4) The effects of predation.
5) Mortality rates by age groups.
6) Diseases and parasites of the Big Bend gambusia.
7) This species' habitat requirements.
The plan calls for monitoring the Big Bend gambusia population
and its habitat, and provides specific management recommendations for
Springs 1 and 4. It recommends that:
1) Spring 4 be restocked with G. gaigei from a captive propagation
program.
Management Practices - 2 (DRAFT) - Management Practices
Species GAMBUSIA, BIG BEND
Species Id ESIS252004
Date 14 MAR 96
2) That campground impacts be minimized through limiting human access,
habitat rehabilitation, and controlling development.
3) That G. affinis (a introduced exotic) be eradicated from the
campground area.
4) The campground should be irrigated from wells rather than directly
from the Rio Grande River so as to eliminate irrigation as a potential
vehicle for immigration of more mosquitofish (G. affinis).
5) A captive population should be maintained at Dexter NFH for
further reintroductions, research, and to provide a reserve gene pool
in the event of a catastrophy.
The Big Bend gambusia should be provided the full protection
mandated by Federal and Texas laws. Finally, public awareness of the
uniqueness to this species should be increased through the preparation
of pamphlets, interpretive programs, and news releases.
Management Practices - 3 (DRAFT) - References
Species GAMBUSIA, BIG BEND
Species Id ESIS252004
Date 14 MAR 96
References
***** REFERENCES FOR ALL NARRATIVES EXCEPT N-OCCURRENCE *****
01 Hubbs, C.L. 1929. Studies of the fishes of the order
Cyprinodontes VIII. Gambusia gaigei, a new species from the Rio
Grande. Occ. Pap. Mus. Zool. Univ. Mich. 198:1-11.
02 Hubbs, C.L. and V.G. Springer. 1957. A revision of Gambusia
nobilis species group, with descriptions of three new species, and
notes on their variation, ecology, and evolution. TX J. Sci.
9:279-372.
03 Rosen, D.E. and R.M. Bailey. 1963. The Poeciliid fishes
(Cyprinodontiformes), their structure, zoogeography, and
systematics. Bull. Amer. Mus. Nat. Hist. 126:1-176.
04 Rivas, L.R. 1963. Subgenera and species groups in the Poeciliid
fish genus Gambusia Poey. Copeia 1963:331-347.
05 Peden, A.E. 1973. Variation in anal spot expression of gambusine
females and its effect on male courtship. Copeia 1973:250-263.
06 Alvarez del Villar, J. 1970. Peces Mexicanos (claves). Serie
investigacion pesquera, estudia No. 1 166 pp.
07 Miller, R.R. 1978. Composition and derivation of the native fish
fauna of the Chihuahuan Desert Region. In: Trans. of the Sym. on
the Biol. Res. of the Chihuahuan Desert Region, U.S.-Mexico. Pp.
365-381. In: R.H. Wauer and D.H. Riskind eds. NPS, GPO,
Washington, DC.
08 U.S. Fish and Wildlife Service. 1984. Big Bend Gambusia (Gambusia
gaigei Hubbs, 1929) Recovery Plan. USFWS, Albuquerque, NM.
32 pp.
09 Deacon, J.E., G. Kobetich, J.D. Williams, S. Contreras et al.
1979. Endangered fishes of North America: Endangered, threatened
or of special concern. Fisheries 4(2):30-44.
10 Hubbs, C.L. and H.J. Broderick. 1963. Current abundance of
Gambusia gaigei, an Endangered fish species. Southwest Nat.
8:46-48.
11 Hubbs, C.L., G. Hoddenbach and C.M. Fleming. 1986. An
enigmatic population of Gambusia gaigei. Southwestern Nat.
12 Hubbs, C.L., J.E. Johnson, and R.H. Wauer. 1977. Habitat
Management Plan for Big Bend Gambusia, Big Bend National Park, TX.
NPS.
13 Moore, G. 1980. Aquifer test of geothermal Spring 4, Rio Grande
Village, Big Bend National Park. NPS, Water Resource Rept. No.
80-8. Southwest Regional Office, Santa Fe, NM 87501.
14 Hubbs, C. and D.T. Mosier. 1986. Fecundity of Gambusia
gaigei, an Endangered species. Copeia.
15 Guillory, V. 1980. Gambusia gaigei Hubbs Big Bend gambusia, Page
540. In: Atlas of North American freshwater fishes. NC Bio. Sur.
Publ. 1980-12.
16 Hubbs, C.L., R. Miller, J. Edwards, K.W. Thompson, E. Marsh, P.
Garrett, G.L. Powell, D.J. Morris, and W. Zerr. 1977. Fishes
inhabiting the Rio Grande, Texas-Mexico, between El Paso and the
Pecos confluence. Pp. 91-97. In: Importance, Preservation and
Management of Riparian Habitat. A symposium. USDA Forest Service,
General Tech. Rept. RM-43. R. Roy Johnson and Dale Jones eds.
References - 1 (DRAFT) - References
Species GAMBUSIA, BIG BEND
Species Id ESIS252004
Date 14 MAR 96
***** REFERENCES FOR N-OCCURRENCE NARRATIVE ONLY *****
01 Hubbs, C.L. 1929. Studies of the fishes of the order
Cyprinodontes VIII. Gambusia gaigei, a new species from the Rio
Grande. Occ. Pap. Mus. Zool. Univ. Mich. 198:1-11.
02 Hubbs, C.L. 1940. Fishes from the Big Bend region of Texas.
Trans. TX Acad. Sci. 23:3-12.
03 Hubbs, C.L. and V.G. Springer. 1957. A revision of Gambusia
nobilis species group, with descriptions of three new species, and
notes on their variation, ecology, and evolution. TX J. Sci.
9:279-372.
04 Hubbs, C.L. and H.J. Broderick. 1963. Current abundance of
Gambusia gaigei, an Endangered fish species. Southwest Nat.
8:46-48.
05 Hubbs, C.L., J.E. Johnson and R.H. Wauer. 1977. Habitat
Management Plan for Big Bend Gambusia, Big Bend National Park, TX.
NPS.
06 Hubbs, C.L. and J.G. Williams. 1979. A review of circumstances
affecting the abundance of Gambusia gaigei, and Endangered fish
endemic to Big Bend National Park. Pp. 631-635. In: Proceeding
First Conf. on Sci. Res. in the National Parks, Vol. 1. Robert
Linn (ed).
07 U.S. Fish and Wildlife Service. 1984. Big Bend Gambusia (Gambusia
gaigei Hubbs, 1929) Recovery Plan. USFWS, Albuquerque, NM.
32 pp.
08 Hubbs, C.L., G. Hoddenbach and C.M. Fleming. 1986. An
enigmatic population of Gambusia gaigei. Southwestern Nat.
09 Hubbs, C.L. 1959. High incidence of vertebral deformities in two
natural populations of fishes inhabiting warm springs. Ecology
40:154-155.
10 Guillory, V. 1980. Gambusia gaigei Hubbs Big Bend gambusia, P.
540. In: Atlas of North American freshwater fishes. NC Bio. Sur.
Publ. 1980-12.
References - 2